Revisiting the catalytic CuZ cluster of nitrous oxide (N2O) reductase. Evidence of a bridging inorganic sulfur

Nitrous-oxide reductases (N2OR) catalyze the two-electron reduction of N(2)O to N(2). The crystal structure of N2ORs from Pseudomonas nautica (Pn) and Paracoccus denitrificans (Pd) were solved at resolutions of 2.4 and 1.6 A, respectively. The Pn N2OR structure revealed that the catalytic CuZ center belongs to a new type of metal cluster in which four copper ions are liganded by seven histidine residues. A bridging oxygen moiety and two other hydroxide ligands were proposed to complete the ligation scheme (Brown, K., Tegoni, M., Prudencio, M., Pereira, A. S., Besson, S., Moura, J. J. G., Moura, I., and Cambillau, C. (2000) Nat. Struct. Biol. 7, 191-195). However, in the CuZ cluster, inorganic sulfur chemical determination and the high resolution structure of Pd N2OR identified a bridging inorganic sulfur instead of an oxygen. This result reconciles the novel CuZ cluster with the hitherto puzzling spectroscopic data.     

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.     

The catalytic center in nitrous oxide reductase, CuZ, is a copper-sulfide cluster

The crystal structure of nitrous oxide reductase, the enzyme catalyzing the final step of bacterial denitrification in which nitrous oxide is reduced to dinitrogen, exhibits a novel catalytic site, called Cu(Z). This comprises a cluster of four copper ions bound by seven histidines and three other ligands modeled in the X-ray structure as OH(-) or H(2)O. However, elemental analyses and resonance Raman spectroscopy of isotopically labeled enzyme conclusively demonstrate that Cu(Z) has one acid-labile sulfur ligand. Thus, nitrous oxide reductase contains the first reported biological copper-sulfide cluster.     

Low-spin heme b(3) in the catalytic center of nitric oxide reductase from Pseudomonas nautica

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mo?ssbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mo?ssbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ～ 6 and g ～ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ～ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.     

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.     

Loss of nitrous oxide reductase in Pseudomonas aeruginosa cultured under N2O as determined by rocket immunoelectrophoresis

The mass ratio of nitrous oxide reductase to total protein in the soluble protein fraction of Pseudomonas aeruginosa P2 was highest in cells grown on nitrate, decreased in cells grown on N(2)O following the exhaustion of the initial charge of nitrate, and was nearly zero in cells exposed solely to N(2)O.     

Effects of sulfide and low redox potential on the inhibition of nitrous oxide reduction by acetylene in Pseudomonas nautica

Membrane introduction mass spectrometry was used to investigate the inhibitory effect of acetylene on the nitrous oxide reductase activity of intact cells of Pseudomonas nautica. We studied the effects of the concentrations of nitrate and sulfide, and the redox potential, which have all been implicated in causing a decrease in the inhibitory effects of acetylene during measurements of denitrification in natural environments. There was no evidence that the concentration of nitrate influenced the effect of acetylene. Lowering the redox potential with the reductant Ti(III)-nitrilotriacetate caused a slight alleviation of acetylene inhibition. Much greater effects at the same redox potential were obtained with concentrations of sulfide in the range 1-10 microM.     

An apparently allosteric effect involving N2O with the nitrous oxide reductase from Wolinella succinogenes.

It was shown that kcat for the benzyl viologen cation (BV+)-N2O oxidoreductase activity of nitrous oxide reductase from Wolinella succinogenes was 2-3 times greater at high N2O concentrations than at low. This effect of N2O on kcat exhibited a titration curve implicating a single secondary binding site for N2O with a Kd of 130-200 microM (Km with respect to N2O is about 2.5 microM). This work represents the first evidence of an apparently allosteric kinetic effect among nitrous oxide reductases. Its possible cause is discussed. BV+ was generated in these kinetic studies by addition of sub-stoichiometric amounts of dithionite. This means of reduction proved to be superior to the photochemical generation of BV+ that had been used previously with the enzyme. Mass spectrometric measurements suggested that the M(r) of the subunit of the enzyme is about 95,500 rather than 88,000.     

Importance of denitrifiers lacking the genes encoding the nitrous oxide reductase for N2O emissions from soil

Analyses of the complete genomes of sequenced denitrifying bacteria revealed that approximately 1/3 have a truncated denitrification pathway, lacking the nosZ gene encoding the nitrous oxide reductase. We investigated whether the number of denitrifiers lacking the genetic ability to synthesize the nitrous oxide reductase in soils is important for the proportion of N₂O emitted by denitrification. Serial dilutions of the denitrifying strain Agrobacterium tumefaciens C58 lacking the nosZ gene were inoculated into three different soils to modify the proportion of denitrifiers having the nitrous oxide reductase genes. The potential denitrification and N₂O emissions increased when the size of inoculated C58 population in the soils was in the same range as the indigenous nosZ community. However, in two of the three soils, the increase in potential denitrification in inoculated microcosms compared with the noninoculated microcosms was higher than the increase in N₂O emissions. This suggests that the indigenous denitrifier community was capable of acting as a sink for the N₂O produced by A. tumefaciens. The relative amount of N₂O emitted also increased in two soils with the number of inoculated C58 cells, establishing a direct causal link between the denitrifier community composition and potential N₂O emissions by manipulating the proportion of denitrifiers having the nosZ gene. However, the number of denitrifiers which do not possess a nitrous oxide reductase might not be as important for N₂O emissions in soils having a high N₂O uptake capacity compared with those with lower. In conclusion, we provide a proof of principle that the inability of some denitrifiers to synthesize the nitrous oxide reductase can influence the nature of the denitrification end products, indicating that the extent of the reduction of N₂O to N₂ by the denitrifying community can have a genetic basis.     

Purification and characterization of nitrous oxide reductase from Pseudomonas aeruginosa strain P2

Nitrous oxide reductase, which catalyzes the reduction of N2O to N2, was purified in a largely oxidized form from Pseudomonas aeruginosa strain P2 by a simple anaerobic procedure to yield an enzyme with a peptide purity of 95-98%. For the native (dimeric) enzyme, Mr = 120,000 and for the denatured subunit, Mr = 73,000. The enzyme contained four Cu atoms/subunit, was purple in color, and exhibited a broad absorption band at 550 nm with an extinction coefficient of about 11,000 M-1 x cm-1 referenced to the dimer. It was nearly inactive as prepared but could be activated by incubation with 2-(N-cyclohexylamino)ethane sulfonate buffer, pH 10, to specific activities as high as 27 mumol of N2O x min-1 x mg-1.Km for N2O and benzyl viologen radical cation was about 2 and 4 microM, respectively, both before and after enzyme activation. Activation increased the t1/2 for turnover-dependent inactivation from about 30 s to 5-10 min. Reduction of the enzyme by dithionite was kinetically biphasic and resulted in the loss of the 550-nm band and ultimate appearance of a 670-nm band. Isoelectric focusing revealed five components with pI values from 5.2 to 5.7. The pI values did not change following activation. The copper CD spectrum of the enzyme as prepared was different from that for the activated enzyme, whereas those for the enzyme after exposure to air and the activated enzyme were similar. Because the activated enzyme is a mixture of activated and inactive species, the specific activity of the activated species must be substantially greater than the observed value. Molecular heterogeneity may also explain the decreased optical absorbance and CD amplitude that resulted from the activation process. The data overall reinforce the view that the absorption spectrum of nitrous oxide reductase is not a good predictor of absolute activity.     

A novel type of catalytic copper cluster in nitrous oxide reductase

Nitrous oxide (N20) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N20 elimination from the biosphere, N20 reductases catalyze the two-electron reduction of N20 to N2. These 2 x 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N20 reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 A. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N20 binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.     

Symbiotic Bradyrhizobium japonicum reduces N2O surrounding the soybean root system via nitrous oxide reductase

N(2)O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N(2)O uptake and conversion of (15)N-N(2)O into (15)N-N(2). Free-living cells of USDA110 showed N(2)O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N(2)O reductase activity. When detached soybean nodules formed with USDA110 were fed with (15)N-N(2)O, they rapidly emitted (15)N-N(2) outside the nodules at a ratio of 98.5% of (15)N-N(2)O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N(2)O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N(2)O (0.34 ppm). These results indicate that the conversion of N(2)O to N(2) depends exclusively on the respiratory N(2)O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N(2)O.     

Enhanced reduction of nitrous oxide by Pseudomonas denitrificans with perfluorocarbons

Nitrous oxide reduction and nitrogen production by Pseudomonas denitrificans, as well as culture growth rates all increased 2-3 fold when cultured in the presence of perfluorocarbon emulsions (10% v/v) as compared to control cultures grown in the absence of perfluorocarbons. Initial nitrous oxide concentrations for consecutive experiments were 0.7 and 1.2 mM respectively.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Identification of nitric oxide and nitrous oxide as products of nitrite reduction by Pseudomonas cytochrome oxidase (nitrate reductase)

The cytosol fraction of rat adrenocortical tissue contains comparatively high levels of two prostaglandin metabolizing enzymes. The first, prostaglandin-9-ketoreductase, utilizes NADPH more effectively than NADH as cofactor, is inhibited by NADP, and exhibits an apparent K_m of 304 μM for PGE₁. 15-hydroxyprostaglandin dehydrogenase, tentatively identified as the type II NADP-dependent isozyme, is inhibited by NADPH but not NADH, and exhibits an apparent K_m of 157 μM when PGE₁ is used as substrate. Changes in specific activities of the two enzymes following ACTH, hypophysectomy, or dexamethasone treatment are inconclusive in defining a chronic regulatory role for adrenocorticotropin.     

Electron transfer complex between nitrous oxide reductase and cytochrome c552 from Pseudomonas nautica: kinetic, nuclear magnetic resonance, and docking studies

The multicopper enzyme nitrous oxide reductase (N 2OR) catalyzes the final step of denitrification, the two-electron reduction of N 2O to N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c 552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c 552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c 552 concentration dependence, we estimate the following kinetic parameters: K m c 552 = 50.2 +/- 9.0 muM and V max c 552 = 1.8 +/- 0.6 units/mg. The N 2O concentration dependence indicates a K mN 2 O of 14.0 +/- 2.9 muM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c 552 is used as the electron donor (p K a = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by (1)H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center.     

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.     

A Pseudomonas stutzeri outer membrane protein inserts copper into N2O reductase

Among a set of frameshift mutagen (ICR-191; Polysciences, Inc.)-induced mutations that confer inability to grow anaerobically with N2O as the sole electron acceptor, one class was found that produced an inactive N2O reductase which lacked copper. All of these mutant strains failed to produce a 61,000-Mr protein located in the outer membrane. This protein, termed NosA, seems not to be responsible for bringing copper into the cell because the mutant strains and their parent were similarly sensitive to the copper content of the growth medium and no intermediate copper concentration in the medium permitted the mutant strains (nosA) to grow anaerobically with N2O as the sole electron acceptor. We conclude that NosA is necessary to insert copper into N2O reductase or to maintain it there.