Nitrosocyanin, a red cupredoxin-like protein from Nitrosomonas europaea

Nitrosocyanin (NC), a soluble, red Cu protein isolated from the ammonia-oxidizing autotrophic bacterium Nitrosomonas europaea, is shown to be a homo-oligomer of 12 kDa Cu-containing monomers. Oligonucleotides based on the amino acid sequence of the N-terminus and of the C-terminal tryptic peptide were used to sequence the gene by PCR. The translated protein sequence was significantly homologous with the mononuclear cupredoxins such as plastocyanin, azurin, or rusticyanin, the type 1 copper-binding region of nitrite reductase, and the binuclear CuA binding region of N(2)O reductase or cytochrome oxidase. The gene for NC contains a leader sequence indicating a periplasmic location. Optical bands for the red Cu center at 280, 390, 500, and 720 nm have extinction coefficients of 13.9, 7.0, 2.2, and 0.9 mM(-1), respectively. The reduction potential of NC (85 mV vs SHE) is much lower than those for known cupredoxins. Sequence alignments with homologous blue copper proteins suggested copper ligation by Cys95, His98, His103, and Glu60. Ligation by these residues (and a water), a trimeric protein structure, and a cupredoxin beta-barrel fold have been established by X-ray crystallography of the protein [Lieberman, R. L., Arciero, D. M., Hooper, A. B., and Rosenzweig, A. C. (2001) Biochemistry 40, 5674-5681]. EPR spectra of the red copper center indicated a Cu(II) species with a g(parallel) of 2.25 and an A(parallel) of 13.8 mT (144 x 10(-4) cm(-1)), typical of Cu in a type 2 copper environment. NC is the first example of a type 2 copper center in a cupredoxin fold. The open coordination site and type 2 copper suggest a possible catalytic rather than electron transfer function.     

Crystal structure of a novel red copper protein from Nitrosomonas europaea

Nitrosocyanin (NC) is a mononuclear red copper protein isolated from the ammonia oxidizing bacterium Nitrosomonas europaea. Although NC exhibits some sequence homology to classic blue copper proteins, its spectroscopic and electrochemical properties are drastically different. The 1.65 A resolution crystal structure of oxidized NC reveals an unprecedented trimer of single domain cupredoxins. Each copper center is partially covered by an unusual extended beta-hairpin structure from an adjacent monomer. The copper ion is coordinated by His 98, His 103, Cys 95, a single side chain oxygen of Glu 60, and a solvent molecule. In the 2.3 A resolution structure of reduced NC, His 98 shifts away from the copper ion, and the solvent molecule is not observed. The arrangement of these ligands renders the coordination geometry of the NC red copper center distinct from that of blue copper centers. In particular, the red copper center has a higher coordination number and lacks the long Cu-S(Met) and short Cu-S(Cys) bond distances characteristic of blue copper. Moreover, the red copper center is square pyramidal whereas blue copper is typically distorted tetrahedral. Analysis of the NC structure provides insight into possible functions of this new type of biological copper center.     

Characterization of Nitrosomonas europaea immobilized in calcium alginate

Nitrosomonas europaea cells have been immobilized in calcium alginate and the resulting preparation was used as a biocatalyst for the oxidation of NH⁺₄ to NO^?₂. Characterization of this immobilized biocatalyst was done according to the guidelines recommended by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The most important indications obtained from the results are: (a) at low concentrations of substrate, either ammonium ions or oxygen, diffusion limitation will play a role; (b) inhibition by nitrite ions accumulating in the support is not rapidly controlling the efficiency of the immobilized cells; (c) accumulation of hydrogen ions is a rate-limiting factor, especially in unbuffered solutions; (d) the activity of immobilized N. europaea can increase as a result of growth in the support under conditions which would cause washout of free cells. This last result shows the potential of immobilized N. europaea for nitrification of wastewater. The development of a system applying a cheaper and more stable support is, however, a prerequisite for this application.     

Cytochrome aa3 from Nitrosomonas europaea

Cytochrome c oxidase has been purified from the ammonia oxidizing chemoautotroph Nitrosomonas europaea by ion-exchange chromatography in the presence of Triton X-100. The enzyme has absorption maxima at 420 and 592 nm in the resting state and at 444 and 598 nm in the dithionite-reduced form; optical extinction coefficient (598 nm minus 640 nm) = 21.9 cm-1 nM-1. The enzyme has approximately 11 nmol of heme a and approximately 11 nmol of copper per mg of protein (Lowry procedure). There appear to be three subunits (approximate molecular weights 50,800, 38,400, and 35,500), two heme groups (a and a3), and two copper atoms per minimal unit. The EPR spectra of the resting and partially reduced enzyme are remarkably similar to the corresponding spectra of the mitochondrial cytochrome aa3-type oxidase. Although the enzyme had been previously classified as "cytochrome a1" on the basis of its ferrous alpha absorption maximum (598 nm), its metal content and EPR spectral properties clearly show that it is better classified as a cytochrome aa3. Neither the data reported here nor a review of the literature supports the existence of cytochrome a1 as an entity discrete from cytochrome aa3. The purified enzyme is reduced rapidly by ferrous horse heart cytochrome c or cytochrome c-554 from N. europaea, but not with cytochrome c-552 from N. europaea. The identity of the natural electron donor is as yet unestablished. With horse heart cytochrome c as electron donor, the purified enzyme could account for a significant portion of the terminal oxidase activity in vivo.     

Anaerobic metabolism of Nitrosomonas europaea

Nitrosomonas europaea is capable of maintaining an anaerobic metabolism, using pyruvate as an electron donor and nitrite as an electron acceptor; utilization of nitrite depends upon supply of both pyruvate and ammonia. The role of ammonia in this reaction was not determined. N europaea also assimilates CO₂ anaerobically into cell material in the presence of nitrite (0.5–1.0 mM), pyruvate and ammonia. This reaction was partially inhibited by nitrite which apparently competed with CO₂ for reducing power. Anaerobic nitrite respiration is sensitive to ionophores, FCCP being the most effective.Non-standard-abbreviations TCA trichloroacetic acid - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazon     

Purification and properties of peroxidase from Nitrosomonas europaea

Peroxidase from the obligate chemosynthetic bacterium Nitrosomonas europaea was purified 1,500-fold, and its properties were examined. The enzyme had a molecular weight of 53,000 and exhibited characteristic absorption maxima at 410, 524, and 558 mmu. The optimal pH and temperature were 7.5 and 44 C, respectively. The peroxidase reaction had an energy of activation of 5,850 cal/mole and required a primary substrate (H(2)O(2)) concentration of 7 x 10(-6)m to proceed at half maximal velocity (K(m)). Reduced cytochrome, c,p-phenylenediamine and pyrogallol acted as hydrogen donors to the purified peroxidase-H(2)O(2) complex. Conditions most suitable for the chemical oxidation of ammonium by H(2)O(2) were determined. The reaction was rapid and produced nitrite but no nitrate. Hydroxylamine was not detected as an intermediate, but it could substitute for ammonium in the system. Neither the rate nor the extent of these reactions was influenced by purified peroxidase, and no evidence was obtained to support a conclusion that the enzyme performs a vital role in the transformation of ammonium to nitrite by N. europaea.     

Quaternary structure of the hydroxylamine oxidoreductase from Nitrosomonas europaea

The hydroxylamine oxidoreductase from Nitrosomonas europaea was prepared to apparent electrophoretic homogeneity. Electron microscopy of negatively stained preparations of the sample revealed an overall diameter of about 8.8 nm of the enzyme particle. The native structure was determined as a tetrahedron-like assembly of identical subunits exhibiting four protein masses.Abbreviations ESI Electron spectroscopic imaging - HAO Hydroxylamine oxidoreductase     

Nitrite as a stimulus for ammonia-starved Nitrosomonas europaea

Ammonia-starved cells of Nitrosomonas europaea are able to preserve a high level of ammonia-oxidizing activity in the absence of ammonium. However, when the nitrite-oxidizing cells that form part of the natural nitrifying community do not keep pace with the ammonia-oxidizing cells, nitrite accumulates and may subsequently inhibit ammonia oxidation. The maintenance of a high ammonia-oxidizing capacity during starvation is then nullified. In this study we demonstrated that cells of N. europaea starved for ammonia were not sensitive to nitrite, either when they were starved in the presence of nitrite or when nitrite was supplied simultaneously with fresh ammonium. In the latter case, the initial ammonia-oxidizing activity of starved cells was stimulated at least fivefold.     

Cometabolism of trihalomethanes by Nitrosomonas europaea

The ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) was shown to degrade low concentrations (50 to 800 mug/liter) of the four trihalomethanes (trichloromethane [TCM], or chloroform; bromodichloromethane [BDCM]; dibromochloromethane [DBCM]; and tribromomethane [TBM], or bromoform) commonly found in treated drinking water. Individual trihalomethane (THM) rate constants (k1THM) increased with increasing THM bromine substitution, with TBM > DBCM > BDCM > TCM (0.23, 0.20, 0.15, and 0.10 liters/mg/day, respectively). Degradation kinetics were best described by a reductant model that accounted for two limiting reactants, THMs and ammonia-nitrogen (NH3-N). A decrease in the temperature resulted in a decrease in both ammonia and THM degradation rates with ammonia rates affected to a greater extent than THM degradation rates. Similarly to the THM degradation rates, product toxicity, measured by transformation capacity (Tc), increased with increasing THM bromine substitution. Because both the rate constants and product toxicities increase with increasing THM bromine substitution, a water's THM speciation will be an important consideration for process implementation during drinking water treatment. Even though a given water sample may be kinetically favored based on THM speciation, the resulting THM product toxicity may not allow stable treatment process performance.     

Enzyme Immunoassay Detection of Nitrosomonas europaea

An exploratory effort to selectively detect the presence of a nitrifying bacterium, Nitrosomonas europaea, successfully demonstrated the fundamental utility of an enzyme-based immunoassay protocol. The applied polyclonal antibody test seemingly offered a marked improvement over the available analytical options, including plating, activity, and fluorescence immunoassay techniques. Following an initial purification step to enhance overall specificity, this procedure had an apparent lower limit of detection of ~5 × 10⁶ cells per ml. Tests conducted with activated sludge samples exhibited a distinct difference between nitrifying and nonnitrifying mixed liquors, although the highest Nitrosomonas levels observed (i.e., at 1 to 2% of the overall viable cell density) were relatively close to the latter detection boundary.     

Cometabolism of Trihalomethanes by Nitrosomonas europaea

The ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) was shown to degrade low concentrations (50 to 800 μg/liter) of the four trihalomethanes (trichloromethane [TCM], or chloroform; bromodichloromethane [BDCM]; dibromochloromethane [DBCM]; and tribromomethane [TBM], or bromoform) commonly found in treated drinking water. Individual trihalomethane (THM) rate constants () increased with increasing THM bromine substitution, with TBM > DBCM > BDCM > TCM (0.23, 0.20, 0.15, and 0.10 liters/mg/day, respectively). Degradation kinetics were best described by a reductant model that accounted for two limiting reactants, THMs and ammonia-nitrogen (NH₃-N). A decrease in the temperature resulted in a decrease in both ammonia and THM degradation rates with ammonia rates affected to a greater extent than THM degradation rates. Similarly to the THM degradation rates, product toxicity, measured by transformation capacity (T_c), increased with increasing THM bromine substitution. Because both the rate constants and product toxicities increase with increasing THM bromine substitution, a water's THM speciation will be an important consideration for process implementation during drinking water treatment. Even though a given water sample may be kinetically favored based on THM speciation, the resulting THM product toxicity may not allow stable treatment process performance.     

Nitrite as a Stimulus for Ammonia-Starved Nitrosomonas europaea

Ammonia-starved cells of Nitrosomonas europaea are able to preserve a high level of ammonia-oxidizing activity in the absence of ammonium. However, when the nitrite-oxidizing cells that form part of the natural nitrifying community do not keep pace with the ammonia-oxidizing cells, nitrite accumulates and may subsequently inhibit ammonia oxidation. The maintenance of a high ammonia-oxidizing capacity during starvation is then nullified. In this study we demonstrated that cells of N. europaea starved for ammonia were not sensitive to nitrite, either when they were starved in the presence of nitrite or when nitrite was supplied simultaneously with fresh ammonium. In the latter case, the initial ammonia-oxidizing activity of starved cells was stimulated at least fivefold.     

Inhibition of biofilm populations of Nitrosomonas europaea

The influence of surface attachment and growth on inhibition of the ammonia oxidizing bacterium, Nitrosomonas europaea, by nitrapyrin was investigated in liquid culture in the presence and absence of glass slides. Significant attachment to glass slides occurred in the absence of ammonia, but the extent of attachment was not affected by nitrapyrin, nor by previous culture of cells in medium containing nitrapyrin. The presence of glass slides affected neither the specific growth rate of N. europaea, measured by changes in nitrite concentration, nor inhibition by nitrapyrin. Inhibitory effects of nitrapyrin on increases in nitrite concentration and in free cell concentration were similar, but greater effects were observed on changes in attached cell concentration. Established biofilms on glass slides grew at a lower specific growth rate than freely suspended cells. Both biofilm cells, and those detached from the biofilm, were protected from inhibition. A mechanism for protection of biofilm populations is proposed involving reduced sensitivity of slowly growing cells producing extracellular polymeric material. Offprint requests to.: J. I. Prosser.     

Nitrosomonas europaea expresses a nitric oxide reductase during nitrification

In this paper, we report the identification of a norCBQD gene cluster that encodes a functional nitric oxide reductase (Nor) in Nitrosomonas europaea. Disruption of the norB gene resulted in a strongly diminished nitric oxide (NO) consumption by cells and membrane protein fractions, which was restored by the introduction of an intact norCBQD gene cluster in trans. NorB-deficient cells produced amounts of nitrous oxide (N2O) equal to that of wild-type cells. NorCB-dependent activity was present during aerobic growth and was not affected by the inactivation of the putative fnr gene. The findings demonstrate the presence of an alternative site of N2O production in N. europaea.     

Chemolithoorganotrophic growth of Nitrosomonas europaea on fructose

The nitrifying bacterium Nitrosomonas europaea can obtain all its carbon for growth from CO(2) and all its energy and reductant for growth from the oxidation of NH(3) and is considered an obligate chemolithoautotroph. Previous studies have shown that N. europaea can utilize limited amounts of certain organic compounds, including amino acids, pyruvate, and acetate, although no organic compound has been reported to support the growth of N. europaea. The recently completed genomic sequence of N. europaea revealed a potential permease for fructose. With this in mind, we tested if N. europaea could utilize fructose and other compounds as carbon sources to support growth. Cultures were incubated in the presence of fructose or other organic compounds in sealed bottles purged of CO(2). In these cultures, addition of either fructose or pyruvate as the sole carbon source resulted in a two- to threefold increase in optical density and protein content in 3 to 4 days. Studies with [(14)C]fructose showed that >90% of the carbon incorporated by the cells during growth was derived from fructose. Cultures containing mannose, glucose, glycerol, mannitol, citrate, or acetate showed little or no growth. N. europaea was not able to grow with fructose as an energy source, although the presence of fructose did provide an energy benefit to the cells. These results show that N. europaea can be grown in CO(2)-free medium by using fructose and pyruvate as carbon sources and may now be considered a facultative chemolithoorganotroph.