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PubMed1994

Sequence of hcy, a gene encoding cytochrome c-554 from Nitrosomonas europaea.

Cytochrome c-554 (Cyt c-554) was purified from Nitrosomonas europaea. The N-terminal and internal amino acid sequences were determined. A synthetic oligodeoxyribonucleotide primer based on the N-terminal sequence was used to construct a PCR clone. This clone was used to identify genomic DNA fragments containing the gene encoding Cyt c-554. We determined the nucleotide sequence of this gene and named it hcy for hydroxylamine oxidoreductase-linked cytochrome.

Amino Acid Sequence↗

PubMed2005

A rapid and simple respirometric biosensor with immobilized cells of Nitrosomonas europaea for detecting inhibitors of ammonia oxidation.

As obligate chemolithotrophs, ammonia-oxidizing bacteria (AOB) grow very slowly and are known to be extremely sensitive to a wide variety of inhibitors. Since it is generally accepted that inhibition of ammonia oxidation by AOB results in a total failure of nitrogen removal, it is necessary to develop a method to detect inhibitors of ammonia oxidation in wastewater. Since ammonia oxidation accompanies oxygen consumption, ammonia oxidation can be easily evaluated by measuring oxygen consumption rate using a dissolved oxygen (DO) probe. In this study, a rapid and simple respirometric biosensor using the pure culture of Nitrosomonas europaea was developed. N. europaea was cultivated in a continuous fermentor operating at the dilution rate of 0.008 h(-1) to obtain physiologically constant cells and was immobilized onto the dialysis membrane through filtration. DO, determined by the biosensor, started to increase 30 s later after ammonia oxidation inhibitor was fed, and a new steady-state DO was obtained in 10-30 min. For this DO profile, steady-state kinetics was applied to evaluate ammonia oxidation efficiency. The concentration of a toxic compound causing 50% decrease of oxygen-consumption activity (EC50) was determined for different chemicals. The EC50 values obtained with the biosensor (0.018 mg l(-1) for allylthiourea, 0.027 mg l(-1) for thioacetamide, 1.10 mg l(-1) for phenol and 0.0 1mg l(-1) for thiourea) indicated that the developed biosensor was highly sensitive to a variety of the inhibitors. It was also shown that the biosensor is applicable for on-line real time monitoring.

Ammonia↗

PubMed2006

Protonation and inhibition of Nitrosomonas europaea cytochrome c peroxidase observed with protein film voltammetry.

The impact of protonation and inhibitor binding of the diheme cytochrome c peroxidase (CCP) from Nitrosomonas europaea has been examined by the technique of catalytic protein film voltammetry (PFV). Previous efforts have shown that the low-potential heme active site (L) binds substrate and yields electrocatalysis at an pyrolytic graphite edge electrode, with properties evocative of a high-potential intermediate, with E(m)>540mV (vs. normal hydrogen electrode) [A.L. Bradley, S.E. Chobot, D.M. Arciero, A.B. Hooper, S. J. Elliott, J. Biol. Chem. 279 (2004) 13297-13300]. Here we demonstrate through similar experiments that catalytic PFV generates limiting currents which allow for electrochemically-detected enzymology of the Ne CCP: such as the demonstration that pH-dependent Michaelis-Menten constants (K(m) values) reveal a pK(a) value of 6.5 associated with the "ES" complex. Further, the direct electrocatalysis is shown in the presence of known inhibitors (cyanide and azide), indicating that inhibitor binding occurs at L, and shifts the resulting catalytic midpoint potential in a negative direction. Michaelis-Menten treatment of the limiting currents generated in the presence of variable concentrations of inhibitors showed that cyanide behaved as a competitive inhibitor with a K(i) value of 0.15muM; azide revealed a mixed-mode of inhibition. The observed data were found to support a previous model of electrocatalysis, and the role of proton transfer chemistry in the active site is discussed in terms of a structural model.

Catalysis↗

PubMed2005

Improved methods for the cultivation of the chemolithoautotrophic bacterium Nitrosomonas europaea.

The chemolithoautotrophic nitrifier Nitrosomonas europaea is useful for biotechnological processes such as bioremediation of toxic chemicals, as well as playing an essential role in the global nitrogen cycle. To successfully apply this bacterium in biotechnology, large quantities of active cells need to be produced. In this paper we demonstrate improved methods for mass cultivation of N. europaea. A doubling in the final cell density to 30 mg dry wt l(-1) was achieved with a fed-batch culture technique using ammonium bicarbonate or sodium bicarbonate for pH control. The cells displayed high ammonia oxidizing activity in excess of 20 mmol g(-1) dry wt h(-1). Continuous culture under energy limited growth conditions (<0.1 mM residual ammonium) in a bench scale bioreactor using a microfiltration membrane for high cell recycle produced final culture densities greater than 350 mg dry wt l(-1). This system allowed operator controlled culturing of cells under excess substrate as well as energy limited growth conditions with a maximum ammonium oxidation potential in excess of 250 mmol g(-1) dry wt h(-1).

Bacteriological Techniques↗

PubMed2004

Biodegradation of natural and synthetic estrogens by nitrifying activated sludge and ammonia-oxidizing bacterium Nitrosomonas europaea.

This report describes the uses of nitrifying activated sludge (NAS) and ammonia-oxidizing bacterium Nitrosomonas europaea to significantly degrade estrone (E1), 17beta-estradiol (E2), estriol (E3), and 17alpha-ethynylestradiol (EE2). Using NAS, the degradation of estrogens obeyed first-order reaction kinetics with degradation rate constants of 0.056 h(-1) for E1, 1.3 h(-1) for E2, 0.030 h(-1) for E3, and 0.035 h(-1) for EE2, indicating that E2 was most easily degraded. Then, we confirmed that E2 was degraded via E1 by NAS. With/without the ammonia oxidation inhibitor, it was observed that ammonia-oxidizing bacteria in conjunction with other microorganisms in NAS degraded estrogens. Using N. europaea, the degradation of estrogens reasonably obeyed zero-order reaction kinetics, and no remarkable difference is present among the four estrogens degradation rates and it was found that E1 was not detected during E2 degradation period. We suggested that E2 was degraded to E1 in NAS could be caused by other heterotrophic bacteria, not by ammonia-oxidizing bacteria.

Ammonia↗

PubMed1998

Primary sequence and solution conformation of ferrocytochrome c-552 from Nitrosomonas europaea.

Cytochrome c-552 from Nitrosomonas europaea is a 9.1-kDa monoheme protein that is a member of the bacterial cytochrome c-551 family. The gene encoding for c-552 has been cloned and sequenced and the primary sequence of the product deduced. Proton resonance assignments were made for all main-chain and most side-chain protons in the diamagnetic, reduced form by two-dimensional NMR techniques. Distance constraints (1056) were determined from nuclear Overhauser enhancements, and torsion angle constraints (88) were determined from scalar coupling estimates. Solution conformations for the protein were computed by the hybrid distance geometry-simulated annealing approach. For 20 computed structures, the root mean squared deviation from the average position of equivalent atoms was 0.84 A (sigma = 0.12) for backbone atoms over all residues. Analysis by residue revealed there were three regions clearly less well defined than the rest of the protein: the first two residues at the N-terminus, the last two at the C-terminus, and a loop region from residues 34 to 40. Omitting these regions from the comparison, the root mean squared deviation was 0.61 A (sigma = 0.13) for backbone atoms, 0.86 A (sigma = 0.12) for all associated heavy atoms, and 0. 43 A (sigma = 0.17) for the heme group. The global folding of the protein is consistent with others in the c-551 family. A deletion at the N-terminus relative to other family members had no impact on the global folding, whereas an insertion at residue 65 did affect the way the polypeptide packs against the methionine-ligated side of the heme. The effects of specific substitutions will be discussed. The structure of c-552 serves to delineate essential features of the c-551 family.

Amino Acid Sequence↗

PubMed1996

Evidence for an iron center in the ammonia monooxygenase from Nitrosomonas europaea.

Binding of the ligand, nitric oxide, in the presence of reductant was used to identify a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex, and a g= 2.03 copper or iron signal in membranes of the ammonia-oxidizing bacterium, Nitrosomonas europaea. The same ferrous S = 3/2 signal is thought to be a component of the membrane-associated methane monooxygenase (pMMO) of Methylococcus capsulatus Bath, since it is seen in the membrane fraction of cells expressing pMMO and in the purified enzyme, but not in the membrane fraction of cells expressing the soluble MMO [Zahn, J.A. and DiSpirito, A.A. (1996) J. Bacteriol. 178, 1018-1029]. Treatment of resting membranes or cells of N. europaea with nitrapyrin, 2-chloro,6-trichloromethylpyridine, resulted in the increase in magnitude of a g = 6, high-spin ferric iron signal. In the presence of NO and reductant, nitrapyrin prevented the formation of the S = 3/2 nitrosyl-iron complex while increasing the intensity of the g = 6 signal. Nitrapyrin is a specific inhibitor of, and is reduced by, the ammonia monoxygenase (AMO) [Bédard, C. and Knowles, R. (1989) Microbiol. Rev. 53, 68-83]. Taken together the data suggest that iron capable of forming the S = 3/2 complex is a catalytic component of AMO of N. europaea, possibly a part of the oxygen-activating center. Inactivation of the membrane-associated AMO with acetylene did not diminish the S = 3/2 nitrosyl-iron signal, the g = 6 signal, or the g = 6 signal.

Acetylene↗

PubMed1997

Evidence for a crosslink between c-heme and a lysine residue in cytochrome P460 of Nitrosomonas europaea.

Cytochrome P460 and hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea catalyze the oxidation of hydroxylamine. Cytochrome P460 contains an unidentified heme-like chromophore whose distinctive spectroscopic properties are similar to those for the P460 heme found in HAO. The heme P460 of HAO has previously been shown by protein chemistry and NMR structural analysis to be a c-heme with an additional covalent crosslink between the C2 ring carbon of a tyrosine residue of the polypeptide chain and a meso carbon of the porphyrin [Arciero, D.M. et al. (1993) Biochemistry 32, 9370-9378]. The recent determination of the gene sequence for cytochrome P460 [Bergmann, D.J. and Hooper, A.B. (1994) FEBS Lett. 353, 324-326] indicates that the heme in this protein also possesses a c-heme binding site and provides the basis for determining whether an HAO-like crosslink exists to the porphyrin. Sequence analysis of a purified heme-containing tryptic chromopeptide from cytochrome P460 revealed two predominant amino acid residues per cycle. Two peptides present in the chromopeptide with the sequences NLPTAEXAAXHK and DGTVTVXELVSV. Comparison of the data to the gene sequence for the protein revealed that the gaps in the first peptide (indicated by X's) code for C residues, confirming the prediction of a c-heme binding motif. The gap in the sequence in the second peptide at cycle 7 is predicted by the gene sequence to be a K. The results suggest that the lysine residue is crosslinked in some manner to the porphyrin macrocycle, possibly mimicking the tyrosine crosslink found for the heme P460 of HAO. While a common role for the crosslinked residues in HAO and cytochrome P460 is difficult to ascertain due to the dissimilarities in side chain structure, it may be related to the similar pKa values for lysine and tyrosine.

Chromatography, Gel↗

PubMed1998

Kinetic characterization of the inactivation of ammonia monooxygenase in Nitrosomonas europaea by alkyne, aniline and cyclopropane derivatives.

The kinetic mechanisms of seven inactivators of ammonia oxidation activity in cells of the nitrifying bacterium, Nitrosomonas europaea were investigated. The effects of the inactivators were specific for ammonia monooxygenase (AMO) which oxidizes ammonia to hydroxylamine. The aniline derivatives, 1,3-phenylenediamine and p-anisidine, were potent inactivators of AMO while other derivatives were ineffective as inactivators. Two cyclopropane derivatives, 1, 2-dimethylcyclopropane and cyclopropyl bromide, were inactivators while cyclopropane was not an inactivator. The mechanisms of three alkynes, 1-hexyne, 3-hexyne, and acetylene, were also examined. For all seven compounds, the inactivation of AMO was irreversible, time-dependent, first-order, and dependent on catalytic turnover. Saturation of the rate of inactivation was indicated for p-anisidine (kinact=2.85 min-1; KI=1.0 mM) and cyclopropyl bromide (kinact=4.4 min-1; KI=97 microM), but not for any of the remaining five inactivators, including acetylene. Ammonia slowed the rate of inactivation for acetylene and cyclopropyl bromide, but enhanced the rate of inactivation for the remaining inactivators. All seven compounds appear to be mechanism-based inactivators of AMO.

Ammonia↗

PubMed1995

Reaction with cyanide of hydroxylamine oxidoreductase of Nitrosomonas europaea.

Hydroxylamine oxidoreductase (HAO) catalyzes the reaction NH2OH+H2O-->HNO2+4e- + 4H+, a step in the energy-generating oxidation of ammonia to nitrite by the bacterium Nitrosomonas europaea. Each subunit of HAO contains 7 c-hemes and 1 heme P460. The latter, c-heme cross-linked from a methylene carbon to the ring of a protein tyrosine, forms part of the active site. The iron of heme P460 is probably linked by a bridging ligand to the iron of a c-heme. Here, the reaction of cyanide with ferric HAO was studied by optical, transient, and steady state kinetic techniques. The molecules, F-, Cl-, Br-, N3-, SCN-, and OCN- did not react with HAO. A single molecule of cyanide bound with high affinity to heme P460 of HAO. The optical and kinetic characteristics of formation of the monocyano complex of HAO resembled those of cyanide derivatives of other heme proteins. Cyanide, in the monocyano complex, was a noncompetitive inhibitor and remained bound during turnover. HAO was found in two forms. The most common form, HAO-A, formed only the monocyano derivative of heme P460, whereas the other, HAO-B, formed a mono- and dicyano complex. The optical properties and kinetics of formation of the mono- and dicyano complexes were different enough to easily allow independent analysis. The optical and kinetic characteristics of formation of the monocyano complex of heme P460 of HAO A and B were very similar. The dicyano complex of HAO-B appeared to result from the addition of a second molecule of cyanide to heme P460. The rate of conversion of the monocyano to the dicyano complex was stimulated 100-fold by the binding of substrate. Formation of the monoheme complex inhibited enzyme activity. The kinetic constants for the first-order formation of the monocyano derivative and the inhibition of substrate oxidation (under either transient or steady-state conditions) were different. The apparent discrepancy could be resolved by the hypothesis that HAO is functionally a dimer in which electrons rapidly equilibrate between the c-hemes of each subunit but not between oligomers. The results form the basis for the use of cyanide as a probe of the active site of HAO.

Binding Sites↗

PubMed1995

Suicide inactivation of hydroxylamine oxidoreductase of Nitrosomonas europaea by organohydrazines.

In the presence of a suitable electron acceptor such as mammalian cytochrome c, hydroxylamine oxidoreductase (HAO) from the chemolithotrophic bacterium Nitrosomonas europaea catalyzes the oxidation of hydroxylamine or hydrazine to nitrite or dinitrogen, respectively. Each subunit of HAO contains 7 c-hemes and a chromophore of the active site called heme P460, a c-heme bridged from a methylene carbon to a ring carbon of a tyrosine of the peptide chain. Reaction with either substrate results in reduction of several c-hemes of HAO. The reaction of organohydrazines with HAO was investigated in this work. HAO was inactivated by (phenyl-, (methyl-, or (hydroxyethyl)hydrazine. The process followed first order kinetics and was inhibited by the substrates, hydroxylamine or hydrazine. Complete loss of enzyme activity and absorbancy characteristic of native heme P460 of HAO occurred at a 1:1 ratio of phenylhydrazine and HAO. HAO was covalently derivatized by two molecules of [14C]-phenylhydrazine per subunit. Heme P460 was derivatized with high affinity, and an amino acid residue was derivatized with lower affinity. c-Hemes were not derivatized except for the partial reaction of (hydroxyethyl)hydrazine with one heme. As with hydroxylamine and hydrazine, incubation with organohydrazines resulted in reduction of c-heme of HAO. Derivatized minus native optical difference spectra of ferric or ferrous HAO revealed changes in the optical properties of heme P460 which were generally similar to shifts seen in the reaction of the heme of other hemoproteins with organohydrazines.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1995

NIH shift in the hydroxylation of aromatic compounds by the ammonia-oxidizing bacterium Nitrosomonas europaea. Evidence against an arene oxide intermediate.

The migration of deuterium and hydrogen was observed in the aromatic hydroxylation of specifically deuterated, monosubstituted benzenes catalyzed by ammonia monooxygenase of Nitrosomonas europaea. The phenolic products of the hydroxylation of aromatics containing ortho-/para-directing substituents (F, Cl, Br, I, OH, NH2, CH3, CH2CH3, and OCH3) were primarily para-phenols. In contrast, with aromatics containing meta-directing substituents (NO2 and CN), the phenolic products were a more even mixture of meta-and para-phenols. ortho-Fluorophenol was the only ortho-phenolic product observed. The nature of the products suggested that the reaction involved an enzyme-specific, electrophilic addition to the aromatic ring so as to favor hydroxylation at either the meta- or para-positions. With the fluoro-, chloro-, and bromobenzene substrates, the values for the migration and retention of deuterium during hydroxylation (NIH shift) were nearly identical when the deuterium was either at the site of hydroxylation or at an adjacent site, indicating a possible common intermediate. The values of the NIH shift with the nitrobenzene substrate were significantly lower when the deuterium was at the site of hydroxylation than at an adjacent site, indicating the operation of a direct loss mechanism. The present results suggest that the aromatic hydroxylation involved a radical or carbocation intermediate which decayed, without the formation of an arene oxide, to form phenolic products with the accompanying direct loss of deuterium at the site of hydroxylation or the shift of the deuterium to an adjacent site.

Ammonia↗

PubMed1993

Evidence for the structure of the active site heme P460 in hydroxylamine oxidoreductase of Nitrosomonas.

Hydroxylamine oxidoreductase (HAO) is responsible for the oxidation of hydroxylamine to nitrite in nitrification by Nitrosomonas europaea. It has an alpha n subunit structure and eight covalently bound hemes per subunit. Seven of these have visible spectra indistinguishable from heme c. The eighth, designated as P460, has unusual visible spectroscopic features in the enzyme and in a heme-containing proteolytic fragment. Its structure has not been previously determined. Enzymatic digestions of HAO were performed, and various proteolytic fragments were purified. Mass spectrometry confirmed the presence of authentic heme c in some fragments, that is, iron protoporphyrin IX cross-linked by two thioether bonds to cysteine residues. It was possible to detect the presence of the P460 pigment in some fragments, based upon the sensitivity of this pigment to treatment of the holoenzyme with hydrogen peroxide. A proteolytic fragment produced by sequential digestion with trypsin and pronase was shown to contain heme c and a hydrogen peroxide-sensitive heme with an unusual visible spectrum. This fragment contained two covalently cross-linked peptides. Mass spectrometry and NMR indicated that the P460 heme was iron protoporphyrin IX covalently bonded by two thioether bridges to peptide, but in addition there was a new, third covalent bond between a meso heme carbon and an aromatic ring carbon on a tyrosyl residue. The new covalent bond has been tentatively assigned to the C2 carbon of the tyrosyl ring and the 5-meso heme carbon (IUPAC-IUB tetrapyrrole nomenclature), although this location requires further proof.

Amino Acid Sequence↗

PubMed1991

Resolution of the four hemes of cytochrome c554 from Nitrosomonas europaea by redox potentiometry and optical spectroscopy.

The electrochemical behavior of tetraheme cytochrome c554 from Nitrosomonas europaea has been studied by thin-layer spectroelectrochemistry, cyclic voltammetry, differential pulse voltammetry, and alternating current voltammetry. Three redox couples were detected. Midpoint potentials for the high-m intermediate-, and low-potential couples are +47, -147, and -276 mV, respectively, from the spectroelectrochemical measurements and +50, -120, and -225 mV, respectively, from the voltammetry measurements. A coulometric titration shows that two electrons are taken up by the high-potential couple and one each is taken up by the intermediate- and low-potential couples. Results from the spectroelectrochemical titration at carefully chosen wavelengths indicate that the intermediate- and low-potential couples obey simple Nernstian behavior. The electrochemical behavior of the high-potential couple is apparently not truly Nernstian but is most consistent with two sites exhibiting slight positive cooperativity. Spectral changes associated with the three couples reveal distinctive features in the reduced-minus-oxidized difference spectra. The difference spectrum of the high-potential pair of hemes suggest a mixture of a high-spin heme and a low-spin heme with maxima at 424 and 432 nm. The difference spectrum of the intermediate-potential heme is low spin with a split Soret with maxima at 414 and 424 nm. The difference spectrum of the low-potential heme also shows a split Soret with maxima at 418 and 432 nm.

Cytochrome c Group↗

PubMed1991

Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitrosomonas europaea.

During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Hydroxide↗

PubMed1977

Hydroxylamine oxidoreductase from Nitrosomonas: inactivation by hydrogen peroxide.

Incubation of hydroxylamine oxidoreductase of Nitrosomonas with hydrogen peroxide resulted in the rapid and irreversible loss of the ability to catalyze the dehydrogenation of hydroxylamine in the presence of electron acceptors, such as phenazine methosulfate. The rate of the reaction was dependent on the concentration of enzyme and H2O2. Inactivation occurred most rapidly at pH values between 9 and 10. Inactivation of the enzyme by H2O2 did not result in alteration of absorption spectrum of either the oxidized form of the enzyme or dithionite-reduced enzyme cytochromes with alpha maxima in the wavelength range 540-570 nm, indicating that those cytochromes were not directly involved in the dehydrogenase step. In contrast to the active enzyme, cytochromes with alpha maxima in the wavelength range 540-570 nm were not reducible by hydroxylamine in the inactivated enzyme. The dithionite-induced absorption maximum at 460 nm (cytochrome P 460), present in the active enzyme, was lost upon inactivation of the enzyme. This is the first direct indication of the involvement of cytochrome P 460 in the action of hydroxylamine oxidoreductase. Protection from inactivation was afforded by (a) substrates for the reduction of enzyme cytochrome, hydrazine, and N-methylhydroxylamine; (b) metal binding agents, KCN, 1,2-dihydroxybenzene-3,5-disulfonate, and hydroxyurea; (c) reductants, o-dianisidine, p-phenylenediamine, hydroquinone, pyrogallol, and dithiothreitol; (d) electron acceptors, phenazine methosulfate, and 2,6-dichlorophenolindophenol; and (e) the singlet oxygen trapping agent, 1,3-diphenylfuran. Scavengers of superoxide anion or hydroxyl radical did not protect the enzyme from inactivation.

Hydrogen Peroxide↗

PubMed2001

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.

Azurin↗

PubMed2002

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.

Amino Acid Sequence↗