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 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.     

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 nos (nitrous oxide reductase) gene cluster from the soil bacterium Achromobacter cycloclastes: Cloning, sequence analysis, and expression

The nitrous oxide (N₂O) reductase (nos) gene cluster from Achromobacter cycloclastes has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ (structural N₂O reductase gene), nosD, nosF, nosY, nosL, and nosX are detected, indicating a genetic organization similar to that of Rhizobium meliloti. To aid homology studies, nosR from R. meliloti has also been sequenced. Comparison of the deduced amino acid sequences with corresponding sequences from other organisms has also allowed structural and functional inferences to be made. The heterologous expression of NosD, NosZ (N₂O reductase), and NosL is also reported. A model of the Cu_A site in N₂O reductase, based on the crystal structure of this site in bovine heart cytochrome c oxidase, is presented. The model suggests that a His residue of the Cu_A domain may be a ligand to the catalytic Cu_Z site. In addition, the origin of the spectroscopically-observed Cys coordination to Cu_Z is discussed in terms of the sequence alignment of seven N₂O reductases.     

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.     

Redox properties of an engineered purple Cu(A) azurin

Purple Cu(A) centers are a class of binuclear, mixed-valence copper complexes found in cytochrome c oxidase and nitrous oxide reductase. An engineered Cu(A) protein was formed by replacing a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Pseudomonas aeruginosa azurin with the corresponding portion of sequence from the Cu(A) center of cytochrome c oxidase from Paracoccus denitrificans [Proc. Natl. Acad. Sci. USA 93 (1996) 461]. Oxidation-reduction midpoint potential (E(m)) values of the Cu(A) azurin of +399+/-10 and +380+/-2mV, respectively, were determined by cyclic voltammetry and spectrochemical titration. An n value of one was obtained, indicating that the redox reaction is cycling between the mixed valence and the fully reduced states. Whereas the E(m) value of native azurin is pH dependent, the E(m) value of Cu(A) azurin is not, as expected for the Cu(A) center. Similarities and differences in the redox properties are discussed in terms of the known crystal structures of Cu(A) centers in cytochrome c oxidase and Cu(A) azurin.     

Nitrous oxide reductase and the 120,000 MW copper protein of N2-producing denitrifying bacteria are different entities

Zumft and Matsubara [1982) FEBS Lett. 148, 107-112) isolated a 120,000 MW copper protein from certain denitrifying bacteria which were capable of producing N2. The presence of this protein correlated with a nutritional requirement of copper for growth on and reduction of N2O by the bacteria. The copper protein was alleged by these workers to be nitrous oxide reductase. However, it is shown that the copper protein and nitrous oxide reductase have different molecular weights and exhibit different behavior upon anion exchange chromatography. The copper protein is therefore not nitrous oxide reductase.     

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)     

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.     

Sulfur ligation in copper enzymes and models

Biological copper-sulfur entities display versatile and unusual coordination chemistry. The role of the sulfur ligation is briefly reviewed through examples from selected copper enzymes and relevant biomimetic models. Copper thiolate complexes are of particular interest because of their key roles in a number of ubiquitous metalloenzymes such as Type I (blue copper proteins) or in the binuclear Cu(A) electrons transfer site found in both cytochrome c oxidase (CcO) and nitrous oxide reductase (N2OR). The possible roles of the S(Met) ligand in monoxygenases are described in relation to recently proposed pathways. Some prospective regarding the biological relevance of disulfide copper ligation and possible radical copper bonds in catalytic cycle are also discussed.     

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.     

Role for zinc(II) in the copper(I) regulated protein CopY.

Despite the importance of copper-thiolate clusters in the regulation of copper metabolism the formation chemistry of these clusters in proteins is not well understood. The number of Cu(I) ions that can be incorporated within a given molecule and their coordination number varies. CopY is a repressor protein from Enterococcus hirae which utilises a copper-thiolate cluster in the regulation of the copper homeostasis genes. Physical, biological assays of purified native reconstituted apoCopY suggest that the formation of a Zn(II)-protein prior to Cu(I) incorporation is necessary to achieve the native Cu(I)-S cluster. In this protein the Zn(II) is readily displaced by the Cu(I). CopY proteins with homologous metal binding motifs are being used to investigate cluster formation stabilisation.     

RNA sequence and the nature of the CuA-binding site in cytochrome c oxidase.

For cytochrome c oxidase subunit II (COXII), DNA and protein sequences suggest that Met-207 (bovine numbering) is conserved in all species except plants. Sequencing of plant mitochondrial COXII mRNAs now indicates that Met-207 is also conserved among plants as a result of a C-to-U type of RNA editing. Considering the strict evolutionary conservation of Met-207 and the homology of COXII to type I (blue) copper proteins and nitrous oxide reductase, we propose a model in which Met-207 is associated with the CuA-binding site (along with Cys-196, Cys-200 and His-204) and plays a role in determining its reduction potential and stability.     

Yeast Cox11, a protein essential for cytochrome c oxidase assembly,is a Cu(I)-binding protein

Cox11 is a protein essential for respiratory growth and has been implicated in the assembly of the Cu(B) site of cytochrome c oxidase. In the present study, we demonstrate that Cox11 is a copper-binding protein. The soluble C-terminal domain of Cox11 forms a dimer that coordinates one Cu(I) per monomer via three thiolate ligands. The two Cu(I) ions in the dimer exist in a binuclear cluster and appear to be ligated by three conserved Cys residues. Mutation of any of these Cys residues reduces Cu(I) binding and confers respiratory incompetence. Cytochrome c oxidase activity is reduced in these mutants. Thus, the residues important for Cu(I) binding correlate with in vivo function, suggesting that Cu(I) binding is important in Cox11 function.     

Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS

The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein. Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.