Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.     

A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri). Evidence from optical, EPR and MCD spectroscopy.

Nitrous oxide reductase (N2OR), Pseudomonas stutzeri, catalyses the 2 electron reduction of nitrous oxide to di-nitrogen. The enzyme has 2 identical subunits (Mr approximately 70,000) of known amino acid sequence and contains approximately 4 Cu ions per subunit. By measurement of the optical absorption, electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectra of the oxidised state, a semi-reduced form and the fully reduced state of the enzyme it is shown that the enzyme contains 2 distinct copper centres of which one is assigned to an electron-transfer function, centre A, and the other to a catalytic site, centre Z. The latter is a binuclear copper centre with at least 1 cysteine ligand and cycles between oxidation levels Cu(II)/Cu(II) and Cu(II)/Cu(I) in the absence of substrate or inhibitors. The state Cu(II)/Cu(I) is enzymatically inactive. The MCD spectra provide evidence for a second form of centre Z, which may be enzymatically active, in the oxidised state of the enzyme. Centre A is structurally similar to that of CuA in bovine and bacterial cytochrome c oxidase and also contains copper ligated by cysteine. This centre may also be a binuclear copper complex.     

Biogenesis of the bacterial respiratory CuA, Cu-S enzyme nitrous oxide reductase

Nitrous oxide reductase (NosZ, EC 1.7.99.6) is the terminal oxidoreductase of a respiratory electron transfer chain that transforms nitrous oxide to dinitrogen. The enzyme carries six Cu atoms. Two are arranged in the mixed-valent binuclear CuA site, and four make up the mu4-sulfide-bridged Cu cluster, CuZ. The biogenesis of a catalytically active NosZ requires auxiliary functions for metal center assembly in the periplasm. Both Tat and Sec pathways share the task to transport the various Nos proteins to their functional sites. Biogenesis of NosZ requires an ABC transporter complex and the periplasmic Cu chaperone NosL. Sustaining whole-cell NosZ function depends on the periplasmic, FAD-containing protein NosX, and the membrane-bound iron-sulfur flavoprotein NosR. Most components with a biogenetic function are now amenable to structural studies.     

The copper site in nitrous oxide reductase

Summary The properties of the novel copper enzyme nitrous oxide reductase from denitrifyingPseudomonas stutzeri are described. Multifrequency electron paramagnetic resonance spectroscopy is used to characterize the various forms of the enzyme. The features observed at 2.4, 3.4, 4.5, 9.31 and 35 GHz are explained by a mixed-valence \s[Cu(1.5)\3. Cu(1.5)\s]S=\12 species with the unpaired electron delocalized between the two Cu nuclei. This site is also present in the catalytically inactive derivative of nitrous oxide reductase which was obtained from a transposon Tn5-induced mutant with defective chromophore biosynthesis. The resemblance of the low-frequency electron paramagnetic resonance spectra to the spectra for the so-called Cu_A of cytochromec oxidase can be taken as a first indication that the Cu_A may have a structural and electronic arrangement similar to the electron-paramagnetic-resonance-detectable copper in nitrous oxide reductase. Results from oxidation/reduction experiments, and from a quantitative determination of sulfhydryl and disulfide residues in the various forms of nitrous oxide reductase, suggest the involvement of the redox-couple cysteine/cystine in the structural organization of the active site of nitrous oxide reductase.     

Anaerobic purification, characterization and preliminary mechanistic study of recombinant nitrous oxide reductase from Achromobacter cycloclastes

An overexpression system for nitrous oxide reductase (N(2)OR), an enzyme that catalyzes the conversion of N(2)O to N(2) and H(2)O, has been developed in Achromobacter cycloclastes. Anaerobically purified A. cycloclastes recombinant N(2)OR (AcN(2)OR) has on average 4.5 Cu and 1.2 S per monomer. Upon reduction by methyl viologen, AcN(2)OR displays a high specific activity: 124 U/mg at 25 degrees C. Anaerobically purified AcN(2)OR displays a unique absorption spectrum. UV-visible and EPR spectra, combined with kinetics studies, indicate that the as-purified form of the enzyme is predominately a mixture of the fully-reduced Cu(Z)=[4Cu(I)] state and the Cu(Z)=[3Cu(I).Cu(II)] state, with the latter readily reducible by reduced forms of viologens. CD spectra of the as-purified AcN(2)OR over a range of pH values reveal perturbations of the protein conformation induced by pH variations, although the principal secondary structure elements are largely unaltered. Further, the activity of AcN(2)OR in D(2)O is significantly decreased compared with that in H(2)O, indicative of a significant solvent isotope effect on N(2)O reduction. These data are in good agreement with conclusions reached in recent studies on the effect of pH on catalysis by N(2)OR [K. Fujita, D.M. Dooley, Inorg. Chem. 46 (2007) 613-615].     

Multiquantum EPR of the mixed valence copper site in nitrous oxide reductase.

This work demonstrates the use of multiquantum EPR to study the magnetic properties of copper complexes and copper proteins. Pure absorption spectra are obtained because of the absence of field modulation. The signal intensity of 3-quantum spectra is proportional to the spin lattice relaxation time T1, while its linewidth in a frequency difference sweep is T1(-1). A change in lineshape for the EPR detectable mixed value [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase is attributed to suppression of the forbidden transitions. The data confirm the unusually fast relaxation time for this site, which requires temperatures of less than 100 K to resolve hyperfine structure. The T1's for the mixed valence [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase are very similar to T1's for the Cua site in cytochrome c oxidase. The similar relaxation properties, together with previous multifrequency EPR results, support the hypothesis that the EPR detectable sites in cytochrome c oxidase and nitrous oxide reductase are mixed valence [Cu(1.5) . . . Cu(1.5)] configurations.     

The NosX and NirX proteins of Paracoccus denitrificans are functional homologues: their role in maturation of nitrous oxide reductase

The nos (nitrous oxide reductase) operon of Paracoccus denitrificans contains a nosX gene homologous to those found in the nos operons of other denitrifiers. NosX is also homologous to NirX, which is so far unique to P. denitrificans. Single mutations of these genes did not result in any apparent phenotype, but a double nosX nirX mutant was unable to reduce nitrous oxide. Promoter-lacZ assays and immunoblotting against nitrous oxide reductase showed that the defect was not due to failure of expression of nosZ, the structural gene for nitrous oxide reductase. Electron paramagnetic resonance spectroscopy showed that nitrous oxide reductase in cells of the double mutant lacked the Cu(A) center. A twin-arginine motif in both NosX and NirX suggests that the NosX proteins are exported to the periplasm via the TAT translocon.     

Nitrous oxide reductase of Flexibacter canadensis: a unique membrane-bound enzyme

Abstract The subcellular distribution of nitrous oxide reductase was studied in the gliding soil bacterium Flexibacter canadensis . Nitrous oxide reductase activity, as measured by the methyl viologen-nitrous oxide oxidoreductase assay, was associated entirely with the membrane fraction of cell-free extracts. The enzyme was liberated from the membranes with use of detergents but not by high-salt concentrations, thus implying that nitrous oxide reductase is an integral membrane protein. The nitrous oxide reductase of F. canadensis is the first reported example of a membrane-bound form of this respiratory enzyme.     

Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.     

Nitrous oxide reductase from Pseudomonas stutzeri. Redox properties and spectroscopic characterization of different forms of the multicopper enzyme

The oxidation-reduction and spectroscopic properties of various forms of nitrous oxide reductase from Pseudomonas stutzeri were investigated. The high-activity form I of the enzyme (purple, 8 Cu, Mr 140,000) was reduced by a large variety of cationic, anionic and photochemically generated agents. The blue form III was the only product found in these experiments under anaerobic conditions. Reductive (dithionite) and oxidative (ferricyanide) titrations showed that the conversion of the purple form I to the blue species III was fully reversible in the absence of dioxygen. Two kinetically different phases of the reaction of form I with a stoichiometric amount of dithionite (1e- -equivalent/Cu) were detected: in the fast phase (seconds), the purple chromophore with lamba max at 540 nm disappeared almost completely, whereas in the slower phase (minutes) the blue species with lambda max around 650 nm was generated. Irrespective of the nature of the reductant the blue species did not react even at large excess of reductant. It was reoxidized by ferricyanide, hydrogen peroxide and nitric oxide. A new, catalytically inactive derivative of nitrous oxide reductase (form V, 2 Cu, Mr 140,000) was isolated from a transposon Tn5-induced mutant with defective chromophore biosynthesis. The pink color of the mutant protein faded almost completely after addition of 0.5e- -equivalent/Cu. In this case no blue species was found, similar to earlier observations for the regenerated, catalytically inactive protein. Varying with the sample and the pH, 50-80% of the total copper of form I was in an electron-paramagnetic-resonance-(EPR)-silent state as compared to 47% in the mutant protein. The broad, featureless EPR signal recorded at 9.32 GHz for the blue, reduced form III of nitrous oxide reductase represented approximately 20% of the total copper. For the blue species no resolution enhancement was achieved at 34 GHz. At this frequency both forms I and V showed similar EPR signals with apparent g-values at 2.16 and 1.99. At 9.32 GHz, form V had an EPR signal with gII at 2.18, AII = 3.55 mT (4 or 5 lines, in contrast to form I) and gI at 2.03. Above 100 K the splitting of the gII region into seven equidistant lines in the EPR signal of the high-activity form I and the hyperfine structure of the perpendicular transition disappeared. Carbon monoxide and nitric oxide, but not nitrous oxide, had marked effects on the spectroscopic properties of the purple form I. Marked effects were also obtained for the exogenous ligands nitrite, azide, cyanate and thiocyanate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron paramagnetic resonance observations on the cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes.

Nitrous oxide reductase from Wolinella succinogenes, an enzyme containing one heme c and four Cu atoms/subunit of Mr = 88,000, was studied by electron paramagnetic resonance (EPR) at 9.2 GHz from 6 to 80 K. In the oxidized state, low spin ferric cytochrome c was observed with gz = 3.10 and an axial Cu resonance was observed with g parallel = 2.17 and g perpendicular = 2.035. No signals were detected at g values greater than 3.10. For the Cu resonance, six hyperfine lines each were observed in the g parallel and g perpendicular regions with average separations of 45.2 and 26.2 gauss, respectively. The hyperfine components are attributed to Cu(I)-Cu(II) S = 1/2 (half-met) centers. Reduction of the enzyme with dithionite caused signals attributable to heme c and Cu to disappear; exposure of that sample to N2O for a few min caused the reappearance of the g = 3.10 component and a new Cu signal with g parallel = 2.17 and g perpendicular = 2.055 that lacked the simple hyperfine components attributed to a single species of half-met center. The enzyme lost no activity as the result of this cycle of reduction and reoxidation. EPR provided no evidence for a Cu-heme interaction. The EPR detectable Cu in the oxidized and reoxidized forms of the enzyme comprised about 23 and 20% of the total Cu, respectively, or about one spin/subunit. The enzyme offers the first example of a nitrous oxide reductase which can have two states of high activity that present very different EPR spectra of Cu. These two states may represent enzyme in two different stages of the catalytic cycle.     

Requirements for Cu(A) and Cu-S center assembly of nitrous oxide reductase deduced from complete periplasmic enzyme maturation in the nondenitrifier Pseudomonas putida

Bacterial nitrous oxide (N(2)O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N(2)O. To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear Cu(A) site in the periplasm. There are seven putative maturation factors, encoded by nosA, nosD, nosF, nosY, nosL, nosX, and sco. We wanted to determine the indispensable proteins by expressing nos genes from Pseudomonas stutzeri in the nondenitrifying organism Pseudomonas putida. An in silico study of denitrifying bacteria revealed that nosL, nosX (or a homologous gene, apbE), and sco, but not nosA, coexist consistently with the N(2)O reductase structural gene and other maturation genes. Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with nosRZ in trans was sufficient to produce catalytically active holo-N(2)O reductase in the nondenitrifying background. We suggest that these obligatory factors are required for Cu-S center assembly. Using a mutational approach with P. stutzeri, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for Cu(A) metallation. Both of these were found to be dispensable elements for N(2)O reductase biosynthesis. Our experimental and in silico data were integrated in a model of N(2)O reductase maturation.     

The tetranuclear copper active site of nitrous oxide reductase: the CuZ center

This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed.     

Biochemical characterization of the purple form of Marinobacter hydrocarbonoclasticus nitrous oxide reductase

Nitrous oxide reductase (N(2)OR) catalyses the final step of the denitrification pathway-the reduction of nitrous oxide to nitrogen. The catalytic centre (CuZ) is a unique tetranuclear copper centre bridged by inorganic sulphur in a tetrahedron arrangement that can have different oxidation states. Previously, Marinobacter hydrocarbonoclasticus N(2)OR was isolated with the CuZ centre as CuZ*, in the [1Cu(2+) : 3Cu(+)] redox state, which is redox inert and requires prolonged incubation under reductive conditions to be activated. In this work, we report, for the first time, the isolation of N(2)OR from M. hydrocarbonoclasticus in the 'purple' form, in which the CuZ centre is in the oxidized [2Cu(2+) : 2Cu(+)] redox state and is redox active. This form of the enzyme was isolated in the presence of oxygen from a microaerobic culture in the presence of nitrate and also from a strictly anaerobic culture. The purple form of the enzyme was biochemically characterized and was shown to be a redox active species, although it is still catalytically non-competent, as its specific activity is lower than that of the activated fully reduced enzyme and comparable with that of the enzyme with the CuZ centre in either the [1Cu(2+) : 3Cu(+)] redox state or in the redox inactive CuZ* state.     

N–O bond cleavage mechanism(s) in nitrous oxide reductase

Quantum chemical calculations of active-site models of nitrous oxide reductase (N(2)OR) have been undertaken to elucidate the mechanism of N-O bond cleavage mediated by the supported tetranuclear Cu(4)S core (Cu(Z)) found in the enzymatic active site. Using either a minimal model previously employed by Gorelsky et al. (J. Am. Chem. Soc. 128:278-290, 2006) or a more extended model including key residue side chains in the active-site second shell, we found two distinct mechanisms. In the first model, N(2)O binds to the fully reduced Cu(Z) in a bent μ-(1,3)-O,N bridging fashion between the Cu(I) and Cu(IV) centers and subsequently extrudes N(2) while generating the corresponding bridged μ-oxo species. In the second model, substrate N(2)O binds loosely to one of the coppers of Cu(Z) in a terminal fashion, i.e., using only the oxygen atom; loss of N(2) generates the same μ-oxo copper core. The free energies of activation predicted for these two alternative pathways are sufficiently close to one another that theory does not provide decisive support for one over the other, posing an interesting problem with respect to experiments that might be designed to distinguish between the two. Effects of nearby residues and active-site water molecules are also explored.     

Purification and some characteristics of nitrous oxide reductase from Paracoccus denitrificans

Nitrous oxide reductase from the denitrifying bacterium Paracoccus denitrificans has been purified very nearly to homogeneity by an anaerobic procedure that results in a product with high specific activity. The enzyme is a dimer of about Mr 144,000 composed of two subunits of apparently equal Mr and contains 4 mol of Cu per mol of subunit. The isoelectric point is 4.3; specific activity at 25 degrees C, pH 7.1, is 122 mumol X min-1 X mg of protein-1; and Km is about 7 microM N2O under the same conditions. The N2O- and O2-oxidized forms of the enzyme had principal absorption bands at 550 and 820 nm; the dithionite-reduced form, at 650 nm. The extinction coefficient at 550 nm for the oxidized enzyme is about 5300 (M subunit)-1 X cm-1. Ferricyanide-oxidized enzyme and enzyme exposed to O2 for a couple of days at 4 degrees C exhibited additional bands at 480, 620, and 780 nm and had very low specific activities. Cu-EPR signals were observed with oxidized and reduced forms of the enzyme with g perpendicular values at 2.042 and 2.055, respectively. The O2-oxidized enzyme had g parallel and A parallel values of about 2.244 and 35 gauss, respectively, based on the observation of four hyperfine lines in the g parallel region. The enzyme may therefore contain at least one Cu atom approximating the "Type 1" class. Spin counts against Cu-EDTA standards suggest that 20-30% of the enzyme-bound Cu is EPR detectable in the O2-oxidized enzyme and 7-15% in the enzyme as prepared and in the reduced enzyme. Much of the Cu thus appears to be EPR silent. Nitrous oxide reductase was observed to undergo turnover-dependent inactivation, and nitrite and fluoride among other anions were found to accelerate this process. In a number of characteristics, the enzyme resembles nitrous oxide reductase recently purified from Pseudomonas perfectomarina and Rhodopseudomonas sphaeroides, particularly the former. Some differences appear related to whether or not purification is carried out entirely under anaerobic conditions.