Electron spin-lattice relaxation of the [Cu(1.5) ... Cu(1.5)] dinuclear copper center in nitrous oxide reductase.

Relaxation times have been obtained with time-domain EPR for the dinuclear mixed valence [CuA(1.5) ... CuA(1.5)[ S = 1/2 center in nitrous oxide reductase, N2OR, from Pseudomonas stutzeri, in the TN5 mutant defective in copper chromophore biosynthesis, in a synthetic mixed valence complex, and in type 1 and 2 copper complexes. Data confirmed that the intrinsic electron spin-lattice relaxation time, T1, for N2OR in the temperature range of 6-25 K is unusually short for copper centers. At best, a twofold increase of T1 from g perpendicular to g parallel was measured. Optimized fits of the saturation-recovery data were obtained using both double-exponential and stretched-exponential functions. The temperature dependence of the spin-lattice relaxation rate of mutant N2OR is about T5.0 with the stretched-exponential model or T3.3 and T3.9 for the model using the sum of two exponentials. These T1s are intrinsic to the mixed valence [CuA(1.5) ... CuA(1.5)] center, and no interaction of the second copper center in wild-type N2OR with the [CuA(1.5) ... CuA(1.5)] center has been observed. The T1 of the mixed valence center of N2OR is not only shorter than for monomeric square planar Cu(II) complexes, but also shorter than for a synthetic mixed valence complex, Cu2(N[CH2CH2NHCH2CH2NHCH2CH2]3N). The short T1 is attributed to the vibrational modes of type 1 copper and/or the metal-metal interaction in [CuA(1.5) ... CuA(1.5)].     

The cupric site in nitrous oxide reductase contains a mixed-valence [Cu(II),Cu(I)] binuclear center: a multifrequency electron paramagnetic resonance investigation

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the assignment of the low field g value at 2.18 consistent with the seven line pattern observed at 9.31 GHz, 10 K. S-band spectra at 20 K are better resolved than the X-band spectra recorded at 10 K. The features observed at 2.4, 3.4, 9.31 and 35 GHz are explained by a mixed-valence [Cu(1.5)..Cu(1.5)] S = 1/2 species with the unpaired electron delocalized between two equivalent Cu nuclei. The resemblance of the N2OR S-band spectra to the spectra for the EPR-detectable Cu of cytochrome c oxidase suggests that the S-band spectrum for cytochrome c oxidase measured below 30 K may also contain hyperfine splittings from two approximately equivalent Cu nuclei.     

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.     

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.     

Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.

The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.     

The nature of CuA in cytochrome c oxidase

Kroneck et al. [(1988) FEBS Lett. 242, 70-74] have recently suggested, on the basis of a comparison with the EPR properties of nitrous oxide reductase, that cytochrome c oxidase contains a mixed-valence binuclear copper site, and that this is responsible for the EPR spectrum generally ascribed to CuA. Here we question this hypothesis in view of a multitude of analytical and spectroscopic data available. We maintain that a mononuclear Cu site with two cysteine sulfur and two imidazole nitrogen atoms as ligands is consistent with the current experimental information on the CuA site.     

Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu-Cu interaction in both enzymes. C-band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well-resolved 7-line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g-values are calculated for gz and gx at four frequencies. No consistent g-values are obtained with the assumption of a 4-line pattern indicative for a mononuclear Cu site.     

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.     

A comparative EPR investigation of the multicopper proteins nitrous-oxide reductase and cytochrome c oxidase.

The multicopper proteins, nitrous-oxide reductase (N2OR) and cytochrome c oxidase (COX), were investigated by EPR spectroscopy at microwave frequencies 2.4-35 GHz. Our results support a Cu-Cu interaction in COX and N2OR. At least 10 lines in the 2.7-GHz, 12 lines in the 4.6-GHz and 14 lines in the 9.2 GHz spectra were resolved for N2OR. Eight copper lines at 2.7 GHz, about nine lines at 4.6 GHz and about six lines at 9.2 GHz were resolved for COX. Simulations of the EPR spectra were consistent with most of the resonances of the multiline spectra, including regions in the center of the spectra where overlap of the three seven-line patterns is proposed. These simulations indicated that Cu-Cu interaction, in a mixed-valence [Cu(1.5) ... Cu(1.5)], S = 1/2 site is consistent with, if not proof of, the unusual spectral features observed for N2OR and COX.     

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.     

Electron spin relaxation of CuA and cytochrome a in cytochrome c oxidase. Comparison to heme, copper, and sulfur radical complexes

The method of continuous saturation has been used to measure the electron spin relaxation parameter T1T2 at temperatures between 10 and 50 K for a variety of S = 1/2 species including: CuA and cytochrome a of cytochrome c oxidase, the type 1 copper in several blue copper proteins, the type 2 copper in laccase, inorganic Cu(II) complexes, sulfur radicals, and low spin heme proteins. The temperature dependence and the magnitude of T1T2 for all of the species examined are accounted for by assuming that the Van Vleck Raman process dominates the electron spin-lattice relaxation. Over the entire temperature range examined, the relaxation of the type 1 coppers in six to seven times faster than that of type 2 copper, inorganic copper, and sulfur radicals, in spite of the similar g-anisotropies of these species. This result may indicate that the coupling of the phonon bath to the spin center is more effective in type 1 coppers than in the other complexes studied. The relaxation of CuA of cytochrome oxidase exhibits an unusual temperature dependence relative to the other copper complexes studied, suggesting that the protein environment of this center is different from that of the other copper centers studied and/or that CuA is influenced by a magnetic dipolar interaction with another, faster-relaxing paramagnetic site in the enzyme. A comparison of the saturation characteristics of the CuA EPR signal in native and partially reduced CO complexes of the enzyme also suggests the existence of such an interaction. The implications of these results with respect to the disposition of the metal centers in cytochrome oxidase are discussed.     

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.     

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.     

Structural features and the reaction mechanism of cytochrome oxidase: iron and copper X-ray absorption fine structure.

X-ray edge absorption of copper and extended fine structure studies of both copper and iron centers have been made of cytochrome oxidase from beef heart, Paracoccus dentrificans, and HB-8 thermophilic bacteria (1-2.5 mM in heme). The desired redox state (fully oxidized, reduced CO, mixed valence formate and CO) in the x-ray beam was controlled by low temperature (-140 degrees C) and was continuously monitored by simultaneous optical spectroscopy and by electron paramagnetic resonance (EPR) monitoring every 30 min of x-ray exposure. The structure of the active site, a cytochrome a3-copper pair in fully oxidized and in mixed valence formate states where they are spin coupled, contains a sulphur bridge with three ligands 2.60 +/- 0.03 A from Fea3 and 2.18 +/- 0.03 A from Cua3. The distance between Fea3 and Cua3 is 3.75 +/- 0.05 A, making the sulphur bond angle 103 degrees reasonable for sp3 sulphur bonding. The Fea3 first shell has four typical heme nitrogens (2.01 +/- 0.03 A) with a proximal nitrogen at 2.14 +/- 0.03 A. The sixth ligand is the bridging sulphur. The Cua3 first shell is identical to oxidized stellacyanin containing two nitrogens and a bridging sulphur. Upon reduction with CO, the active site is identical to reduced stellacyanin for the Cua3 first shell and contains the sulphur that forms the bridge in fully oxidized and mixed valence formate states. The Fea3 first shell is identical to oxyhemoglobin but has CO instead of O2. The other redox centers, Fea and the other "EPR detectable" Cu are not observed in higher shells of Fea3. Fea has six equidistant nitrogens and Cua has one (or two) nitrogens and three (or two) sulphurs with typical distances; these ligands change only slight on reduction. These structures afford the basis for an oxygen reduction mechanism involving oxy- and peroxy intermediates.     

An EPR study of the lineshape of copper in cytochrome c oxidase.

The EPR spectrum of copper in cytochrome c oxidase (EC 1.9.3.1) has been studied between 5 and 220 degreesK, and the spectral parameters have been determined for both forms of EPR-detectable copper by computer simulation methods. Numerical methods have been developed to separate the spectra of intrinsic copper and inactive copper. Evidence is presented to show that inactive copper is probably formed by denaturation. The EPR parameters for intrinsic copper were determined as gx = 1.99, gy = 2.03, gz = 2.185, / Ax(Cu) / = 0.0020 cm-1, / Ay(Cu) / = 0.0025 cm-1, / Az(Cu) / = 0.0030 cm-1. The principal values of the g tensor and the small value of /Az(Cu) / are interpreted in terms of mixing of 3d, 4s, and 4p metal orbitals. A flattened-tetrahedral stereochemistry about Cu2+ with an additional rhombic distrotion is in best agreement with all of the data. The peak-to-peak linewidth is found to be orientation dependent, and is described by a tensor with principal values deltaHx = 45G, deltaHy = 65 G, deltaHz = 85 G. A weak dipolar interaction with a low-spin ferric species stereochemistry for the copper ion is consistent with the electron transport function of the enzyme. Broad EPR signals with a very short spin-lattice relaxation time has been observed near g = 14 and g = 3 at 5 degrees K in oxidized cytochrome oxidase but not in the reduced or denatured enzyme. The possibility that these are due to the "EPR-undetectable" iron and copper is raised.     

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.     

Studies on the origin of the near-infrared (800–900 nm) absorption of cytochrome c oxidase

Data are presented which were collected in the course of the past ten years and bear on the correlation of absorbance at 800 nm and the EPR signal at g = 2 (‘copper signal’) of cytochrome c oxidase in various states of oxidation and ligation. Both EPR and optical reflectance spectra were obtained at low temperature (?170 to ?190°C). For some sets of samples spectra were recorded in the range 500–1100 nm. A particular effort was made to study this correlation with what are called ‘mixed valence’ states (Greenwood, C., Wilson, M.T. and Brunori, M. (1974) Biochem. J. 137, 205–215), when cytochrome a and the EPR-detectable copper are thought to be oxidized and the other components reduced and vice versa. These data show no evidence that the copper component of cytochrome oxidase which has so far not been detected by EPR makes a contribution to the absorption between 800 and 900 nm exceeding 10–15% of the total, which is close to or within the error of the respective measurements. For the various states of the oxidase examined in this work the 700–800 nm region did not appear to be more useful than the 800–900 nm region for determining the state of the EPR-undetectable copper in a reliable way. These conclusions are in agreement with results presented previously from other laboratories concerning the relationship of optical (approx. 800 nm) and EPR spectroscopic (g = 2) data obtained with the enzyme.     

Ligand replacement study at the His120 site of purple CuA azurin

The CuA center is a dinuclear Cu2S2(Cys) electron transfer center found in cytochrome c oxidase and nitrous oxide reductase. In a previous investigation of the equatorial histidine ligands' effect on the reduction potential, electron transfer and spectroscopic properties of the CuA center, His120 in the engineered CuA azurin was mutated to Asn, Asp, and Ala. The identical absorption and EPR spectra of these mutants indicate that a common ligand is bound to the copper center. To identify this replacement ligand, the His120Gly CuA azurin mutant was constructed and purified. Absorption and X-band EPR spectra show that His120Gly is similar to the other His120X (X = Asn, Asp, Ala) mutant proteins. Titrations with chloride, imidazole, and azide suggest that the replacement ligand is not exchangeable with exogenous ligands. The possibility of an internal amino acid acting as the replacement ligand for His120 in the His120X mutant proteins was investigated by analyzing the CuA azurin crystal structure and then converting the likely internal ligand, Asn 119, to Asp, Ser, or Ala in the His120Gly mutant. The double mutants H120G/Asn 119X (X = Asp, Ser, or Ala) displayed UV-Vis absorption and EPR spectra that are identical to His120Gly and the other His120X mutants, indicating that Asn119 is not the internal ligand replacing His120 in the His120X mutant proteins. These results demonstrate the remarkable stability of the dinuclear His120 mutants of CuA azurin.     

Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties.

1. Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20000 Mr could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 +/- 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 +/- 50 units/mg protein or 1088 +/- 15 units/microgram copper. 2. The pure enzyme is characterized by the following optical purity indices: A280/A610 = 25 +/- 0.5, A330/A610 = 0.65 +/- 0.05 and A610/A500 = 7.0 +/- 0.25. The molar absorption coeffient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts of 9700 M-1 cm-1 . 3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, gy = 2.058, gx = 2.036; Az = 5.0 mT, Ay = Ax = 0.5 mT, for the type-2 (non-blue) copper g parallel to = 2.242, g perpendicular = 2.053; A parallel to = 19.0 mT, A perpendicular 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra. 4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductase must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (lambda max ex = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm (delta epsilon = 1000 +/- 50 M-1 cm-1). Corresponding changes in EPR and fluorescence spectra have not been detected.     

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.