Achromobacter Cycloclastes Research Articles

Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.

The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.

Electron-transfer reactions of blue copper protein pseudoazurin from Achromobacter cycloclastes with cytochromes

Electron-transfer rate constants of the single type I Cu protein pseudoazurin from Achromobacter cycloclastes with cytochrome c 551 from Pseudomonas aeruginosa and cytochrome c from bovine heart were determined to be (4.55 ± 0.01) x 10 5 M -1 s -1 and 384 ± 3 M -1 s -1 respectively at pH 7.0 (25°C) by the stopped-flow method, 1 = 0.1 M (NaCI). Using the Marcus equations, corresponding cross-reaction rate constants were calculated using the self-exchange rate constants and compared to the observed rate constants. The cross-reaction rate constants of pseudoazurin with Pseudomonas aeruginosa cytochrome c 551 and bovine heart cytochrome c were calculated to be 1.4 X 10 5 M -1 s -1 and 1.8 X 10 3 M -1 s -1 respectively. The pH effect on the reaction of pseudoazurin with bovine heart cytochrome c was investigated. The obtained kinetic parameters for the cytochrome c oxidation of reduced pseudoazurin estimated to be p K H = 6.5 ± 0.2, k o = 924 ± 77 M -1 s -1 (deprotonated form), and k H = 184 ± 52 M -1 s -1 (protonated form).

Comparison between the oxidized and the reduced structures of pseudoazurin from Achromobacter cycloclastes IAM1013

Comparison between the oxidized and the reduced structures of pseudoazurin from Achromobacter cycloclastes IAM1013

Nitrate- and nitrite-ligated 3,6-bis(imidazolyl)pyridazine-bridged dinuclear copper(II) cations with copper–copper separations similar to that in Achromobacter cycloclastes nitrite reductase

The 4,4′-bipyridine (4,4′-bipy) and 3,6-bis(imidazolyl)pyridazine (bimpydz) bridged dinuclear copper(II)–diethylenetriamine (dien) complexes, [{Cu(dien)}2(µ-diimine)][NO3]4·xH2O (diimine = 4,4′-bipy, x= 2 1; diimine = bimpydz, x= 0 5), [{Cu(dien)}2(µ-diimine)][BF4]4·4MeCN (diimine = 4,4′-bipy 2 or bimpydz 6) and [{Cu(dien)}2(µ-bimpydz)]Cl4·4H2O 4, have been synthesised and characterised. Reaction of the tetrafluoroborates, 2 and 6, with NaNO2 yields the nitrites [{Cu(dien)}2(µ-diimine)][NO2][BF4]3·xMeCN (diimine = 4,4′-bipy, x= 0 3; diimine = bimpydz, x= 0.5 7). Neither the chloride nor the nitrates react with NaNO2. Structural analysis of complexes 5 and 7 has shown that although they are both based on the dinuclear cationic unit, [{Cu(dien)}2(µ-bimpydz)]4+, in 5 the bimpydz bridge adopts a transoid arrangement of imidazole molecules, whereas in 7 it adopts a cisoid arrangement, giving Cu ⋯ Cu separations of 13.28 and 12.88 A, respectively. In 5, the dications are linked by two axially co-ordinated nitrate anions, one strongly bound, the other very weakly bound, to give chains with a ladder-type motif. In 7, the dications are bridged by µ-nitrito-κO : κN moieties to form a chain with helical geometry. The copper(II)–nitrite interaction is novel; the anion bridges the weakly binding axial positions of two square-pyramidal copper atoms using the nitrogen lone pair and the syn lone pair of an oxygen.

Electronic Structure of the Perturbed Blue Copper Site in Nitrite Reductase: Spectroscopic Properties, Bonding, and Implications for the Entatic/Rack State

Low-temperature optical absorption, circular dichroism, magnetic circular dichroism, and sulfur K-edge X-ray absorption spectra have been measured for the green “blue” copper center (type 1) in Achromobacter cycloclastes nitrite reductase. Combined with density functional calculations, the results of these spectroscopies have been used to define the extremely “perturbed” electronic structure of this site relative to that of the prototypical “classic” site found in plastocyanin. Experimentally calibrated density functional calculations have been further used to determine the specific geometric distortions which generate the perturbed electronic structure. These studies indicate that the principal electronic structure changes in nitrite reductase, relative to plastocyanin, are a rotation of the Cu dx2-y2 half-filled, highest occupied molecular orbital (HOMO) and an increase in the ligand field strength at the Cu center. The HOMO rotation increases the pseudo-σ interaction and decreases the π interaction of ...

The Structure of Copper-nitrite Reductase from Achromobacter cycloclastes at Five pH Values, with NO-2 Bound and with Type II Copper Depleted

High resolution x-ray crystallographic structures of nitrite reductase from Achromobacter cycloclastes, undertaken in order to understand the pH optimum of the reaction with nitrite, show that at pH 5.0, 5.4, 6.0, 6.2, and 6.8, no significant changes occur, other than in the occupancy of the type II copper at the active site. An extensive network of hydrogen bonds, both within and between subunits of the trimer, maintains the rigidity of the protein structure. A water occupies a site approximately 1.5 A from the site of the type II copper in the structure of the type II copper-depleted structure (at pH 5.4), again with no other significant changes in structure. In nitrite-soaked crystals, nitrite binds via its oxygens to the type II copper and replaces the water normally bound to the type II copper. The active-site cavity of the protein is distinctly hydrophobic on one side and hydrophilic on the other, providing a possible path for diffusion of the product NO. Asp-98 exhibits thermal parameter values higher than its surroundings, suggesting a role in shuttling the two protons necessary for the overall reaction. The strong structural homology with cupredoxins is described.

Spectroscopic and Electrochemical Studies on Active-site Transitions of the Type 1 Copper Protein Pseudoazurin from Achromobacter cycloclastes

The single type 1 copper protein pseudoazurin from Achromobacter cycloclastes gives reversible electrochemical behavior at a (4-pyridyl)disulfide-modified gold electrode. Measurements carried out at 25.0 degrees C indicate a midpoint reduction potential of E 1/2 = 260 mV versus normal hydrogen electrode at pH 7.0 and a peak-to-peak separation of delta Ep = 59 mV. The diffusion coefficient and heterogeneous electron transfer rate constant are estimated to be 2.23 x 10(-6) cm2 s-1 and 3.7 x 10(-2) cm s-1, respectively. Also, controlled potential electrolysis indicates a 1-electron transfer process and a formal reduction potential of 259 mV versus normal hydrogen electrode for the Cu(II)/Cu(I) couple. The heterogeneous electron transfer rate constant determined at the (4-pyridyl)disulfide-modified gold electrode at pH 4.6 is 6.7 x 10(-3) cm s-1, consistent with a slower process at the positively charged electrode surface. At pH 11.3, UV-visible, EPR, and resonance Raman spectra indicate a conversion of the distorted tetrahedral copper geometry to a trigonal structure. The trigonal form has elongated axial bonding and an axial EPR spectrum. At pH 11.3, the reduction potential is further decreased, and Cu-S bands in resonance Raman spectra at 330-460 cm-1 are shifted to higher energy (approximately 10 cm-1), consistent with a stronger Cu-S bond.

Replacement of Methionine as the Axial Ligand of Achromobacter cycloclastes Cytochrome C554 at High pH Values Revealed by Absorption, EPR and MCD Spectroscopy

Cytochrome c554 from the denitrifying bacterium Achromobacter cycloclastes is a monoheme class II c-type cytochrome with a His-Met axial coordination at neutral pH. The amino acid composition and the N-terminal sequence of the cytochrome have been determined. Subsequent determination of the pH-dependence of the redox potential and examination of the EPR and MCD spectra of ferricytochrome c554 revealed a new form at high pH values made apparent with both spectroscopies. These observations are consistent with the presence of lysine as the axial ligand for which methionine substitutes at high pH values.

Reactivity of Pseudoazurin from Achromobacter cycloclastes with Inorganic Redox Partners and Related NMR and Electrochemical Studies

Reactivity of Pseudoazurin from Achromobacter cycloclastes with Inorganic Redox Partners and Related NMR and Electrochemical Studies

Proton nuclear magnetic resonance assignments and the electron self-exchange rate constant for pseudoazurin from Achromobacter cycloclastes

A partial assignment of proton resonances has been made for Achromobacter cycloclastes pseudoazurin. These include the imidazole ring resonances of the three histidine residues and also the CεH3 resonances of methionines. Some of these assigned resonances have been used to determine the self-exchange rate constant of pseudoazurin by line-broadening measurements on 1 : 1 mixtures of oxidised and reduced protein. The self-exchange rate constant has also been determined using a Marcus analysis of the rate constant for the cross-reaction between pseudoazurin and azurin. Good agreement between the values determined by these two methods is found with a self-exchange rate constants of 2.9 × 103 M–1 s–1 from NMR and 2.7 × 103 M–1 s–1 from the cross-reaction with azurin, both values being at 25 °C, I= 0.100 M. The self-exchange rate constant for pseudoazurin is much smaller than those of most other type 1 blue copper proteins and is quite similar to those found for the higher plant plastocyanins. From the X-ray crystal structure of pseudoazurin it is known that the copper at the active site is co-ordinated by His, Cys, His and Met residues. On the assumption that exchange is via His-81 protruding at the surface of the protein it is likely that the neighbouring basic residues impede the approach of the two proteins in a suitable orientation for this to occur.

Reversible active site protonation and electron-transfer properties of Achromobacter cycloclastes pseudoazurin: comparisons with other type 1 copper proteins

Kinetic and NMR studies on the copper(I) form of A. cycloclasters pseudoazurin provide evidence for an active site protonation (and dissociation) equilibrium process involving His81, pKa 4.9 (average); the three type 1 copper proteins now known to exhibit this behaviour have just two amino acids separating the ligating Cys and His residues in the C-terminal region.

Direct Electrochemistry of Nitrite Reductase from Achromobacter cycloclastes IAM 1013

Abstract The cyclic voltammetry of the nitrite reductase from Achromobacter cycloclastes IAM 1013 exhibited well defined voltammetric response at a di-4-pyridyl disulfide (4-pyds) modified gold electrode in the presence of A. cycloclastes apopseudoazurin. The midpoint potential, E1/2, of the native and type II copper-depleted (T2D) nitrite reductase obtained from the voltammogram were estimated to be 240 mV and 204 mV vs. NHE, respectively. The almost identical current value of the native and T2D reductase suggested that the voltammetric behavior contributed from the type 1 copper site. When nitrite was added into the nitrite reductase solution (pH 7.0), an enhanced sigmoidal cathodic current-potential curve indicating the catalytic regeneration of oxidized nitrite reductase was observed. The rate constant of nitrite reduction and the Michaelis constant, Km, of the native reductase were estimated to be 5 × 102 mol−1 dm3 s−1 and 7 × 10−4 mol dm−3, respectively. The rate constant of the nitrite reduction and the Km value of T2D nitrite reductase were also estimated to be 1 × 102 mol−1 dm3 s−1 and 3 × 10−3 mol dm−3, respectively. These results suggested that the nitrite reduction activity is closely related with type II copper site.

Synthesis and structural and spectroscopic characterization of mononuclear copper nitrosyl complexes: models for nitric oxide adducts of copper proteins and copper-exchanged zeolites

We report the synthesis and characterization of the first examples of well-characterized mononuclear copper nitrosyl complexes, Tp RR' CuNO (Tp RR' =tri(3-R,5-R'-pyrazolyl)hydroborate: 1, R= t-Bu, R'=H; 2, R=R'= Ph). These novel {CuNO} 11 (10 metal d+1 NO π * =11 total electrons) compounds model a possible intermediate in nitrite reduction by the copper nitrite reductase from Achromobacter cycloclastes and provide chemical precedentfor NO coordination to isolated copper sites in proteins and in zeolites

Crystallization and Preliminary X-Ray Studies on Pseudoazurin from Achromobacter cycloclastes IAM10131

New crystals of a blue copper protein, pseudoazurin from denitrifier Achromobacter cycloclastes IAM1013, have been obtained by means of vapor diffusion with ammonium sulfate as a precipitant at pH 6.0 and 4 degrees C. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 56.69(2), b = 61.53(2), and c = 30.20(1) A. The asymmetric unit includes one molecule of pseudoazurin with a Vm value of 2.04 A3/Da. The crystals are so stable against X-ray irradiation that a complete data set up to 1.54 A has been collected using a single native crystal. Solution of the structure was performed by means of the Patterson search techniques, and the current crystallographic R-factor is 17.5% at 3.0 A resolution. Refinement at higher resolution is in progress.

Resonance Raman spectroscopy of the azurin His117Gly mutant. Interconversion of type 1 and type 2 copper sites through exogenous ligands.

The copper center of the Pseudomonas aeruginosa His117Gly azurin mutant is accessible to exogenous ligands through an aperture in its surface created by the removal of the endogenous imidazole ligand. Depending on the exogenous ligand, a surprising variety of type 1 and type 2 copper sites can be obtained that are readily distinguished by electronic, EPR, and resonance Raman (RR) spectroscopy. The RR spectrum of type 1 H117G with exogenous imidazole is nearly identical to that of wild-type azurin, indicating that the trigonal geometry and short Cu-S(Cys) bond of approximately 2.15 A have been maintained. With anionic ligands (e.g., Cl-, Br-, N3-), the RR spectra show increased intensity at 370 and 400 cm-1 and a corresponding decrease in intensity at 410 cm-1, suggesting a lengthening of the Cu-S(Cys) bond as the site achieves a more tetrahedral character. An extreme example is the hydroxide adduct of H117G which is green in color and has optical and RR spectra reminiscent of the tetrahedral type 1 site in Achromobacter cycloclastes nitrite reductase. The fact that the basic RR pattern is little changed in most of the type 1 adducts indicates that the RR spectrum is due primarily to vibrations of the Cu-cysteinate moiety and that its coplanar conformation is conserved. Type 2 H117G proteins are formed by the addition of bidentate exogenous ligands such as histidine and histamine. They have their absorption maxima blue-shifted to 400 nm and their EPR A parallel values increased to approximately 160 x 10(-4) cm-1, both of which are characteristic of tetragonal Cu sites with Cu-S(thiolate) bonds of > 2.25 A. The RR spectra of the type 2 H117G proteins are still dominated by multiple cysteinate-related vibrational modes. However, the vibrational modes with the greatest intensity and Cu-S(Cys) stretching character have shifted approximately 100 cm-1 to lower energy compared to the type 1 sites, consistent with a longer (Cys)S-Cu bond. It is proposed that the tetragonal type 2 character of the bidentate ligand complexes is due to the addition of a fourth strong ligand in the equatorial ligand plane.

Nitric oxide reductase of Achromobacter cycloclastes

Nitric oxide reductase from Achromobacter cycloclastes was solubilized with dodecyl maltoside from membranes and substantially purified by hydroxyapatite column chromatography. Preparations of enzyme had purities estimated to be 65–72% and specific activities of about 3 μmol NO/min per mg protein when measured at 30°C and pH 5.5 using ascorbate/phenazine methosulfate as the reducing system. Preparations lacked nitrite reductase activity. The enzyme consists of two peptides of approx. 38 and 17.5 kDa, and is identified as a cytochrome bc complex for which the mol ratio of cytochrome c to cytochrome b is about 1:1. A heme c containing band of approx. 55 kDa would appear to be a 1:1 complex or compound of the 38 and 17.5 kDa peptides. These and other observations suggest that A. cycloclastes possesses a nitric oxide reductase similar to that previously purified from Pseudomonas stutzeri and Paracoccus denitrificans. In contrast to the cytochrome cd 1-type nitrite reductase of the latter two bacteria, the nitrite reductase of A. cycloclastes is a copper protein. This is the first reported characterization of nitric oxide reductase from a denitrifier with a Cu-containing nitrite reductase, and provides further evidence that the denitrification pathway in such bacteria requires nitric oxide reductase and proceeds by way of NO as an intermediate.

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd1-free background (NirS-) of Pseudomonas stutzeri.

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of "Achromobacter cycloclastes" nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd1 nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d1, indicating that both heterologous hosts synthesized a cytochrome cd1 without the d1-group.

X-ray scattering using synchrotron radiation shows nitrite reductase from Achromobacter xylosoxidans to be a trimer in solution.

We demonstrate here the applicability of X-ray scattering for studying molecular conformation of multimeric proteins in solution by using synchrotron radiation to extend the range of data collection to include medium angles (ca. 3-4 degrees). We have been able to define the solution structure of the dissimilatory nitrite reductase of Achromobacter xylosoxidans (AxNiR), an enzyme for which there are conflicting reports as to the nature of its multimeric structure. Quantitative interpretation of the X-ray scattering profile, based on a modeling study using the high-resolution crystal structure data for the nitrite reductase from the related organism Achromobacter cycloclastes (AcNiR), provides a detailed model for the trimeric structure of AxNiR in solution. Sedimentation equilibrium centrifugation gave an M(r) of 103,000, consistent with such a trimeric structure.

Direct electrochemistry of copper-containing nitrite reductase from Achromobacter cycloclastes IAM 1013

Direct electrochemistry of copper-containing nitrite reductase from Achromobacter cycloclastes IAM 1013

Evidence that the Type 2 copper centers are the site of nitrite reduction by Achromobacter cycloclastes nitrite reductase

Methods have been developed for selective depletion and reconstitution of the Type 2 Cu (non-blue) sites in the nitrite reductase from A. cycloclastes, resulting in preparations ranging from 0.5 to 2.6 Type Cu per trimer; the Type 1 Cu content is invariant at 3.0 per trimer. The activity of the enzyme is directly proportional to the Type 2 content as measured by direct metal determination or by analysis of the EPR spectra. These results indicate that an earlier report that the A. cycloclastes enzyme contains only Type 1 Cu sites is incorrect, and that the Type 2 Cu centers constitute the site at which NO 2 − is reduced. Furthermore, they suggest that other Cu nitrite reductases that are reported to contain only Type 1 Cu sites and exhibit relatively low activity may actually be largely Type 2 Cu-depleted forms of the enzymes.