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A B Hooper

Publications and source records attributed to A B Hooper.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1994

Characteristics of the paramagnetic 1H-NMR spectra of the ferricytochrome c-551 family.

Heme proton resonances have been assigned for ferricytochromes c-551 isolated from four distinct species of bacteria. While the available structure information indicates that the four cytochromes have very similar conformations in solution, including the chirality of the methionine ligand sulfur bond, the chemical shifts of the paramagnetically shifted resonances are surprisingly different, more so than has been previously reported for a homologous series of ferricytochromes. The resonances are contrasted in terms of chemical shift and the temperature dependence of the shift, which gives rise to a very strong anti-Curie effect for some specific protons. Non-methyl heme resonances do display an approximately conserved set of chemical shifts, but the heme methyl groups demonstrate a wide range of values. The 12(1) heme methyl group is always the highest frequency heme methyl, but the relative positions of the other methyl groups may change. The 7(1) heme methyl group always displayed strong anti-Curie behavior, while the 12(1) methyl group displayed normal Curie behavior. The behavior of the other methyl groups was variable. Possible reasons for the range of observations will be discussed. In spite of their NMR differences, all the ferricytochromes c-551 demonstrated comparable electron-transfer rates to a membrane-bound cytochrome reductase system.

Bacterial Proteins↗
PubMed1994

The primary structure of cytochrome P460 of Nitrosomonas europaea: presence of a c-heme binding motif.

Cytochrome P460 and hydroxyamine oxidoreductase of Nitrosomonas europaea both catalyze the oxidation of hydroxylamine and contain a 460 nm-absorbing chromophore. The gene (cyp) encoding cytochrome P460 was cloned and sequenced. The predicted amino acid sequence contains a single c-heme binding motif (CXXCH) near the carboxy-terminus. Cytochrome P460 shows little sequence homology to other c-cytochromes including hydroxyamine oxidoreductase. The presence of a signal peptide and a possible c-heme binding site suggest that the cytochrome P460 of N. europaea is periplasmic.

Amino Acid Sequence↗
PubMed1994

A di-heme cytochrome c peroxidase from Nitrosomonas europaea catalytically active in both the oxidized and half-reduced states.

A di-c-heme containing cytochrome (cytochrome c553 peroxidase) has been isolated from the chemoautotrophic bacterium Nitrosomonas europaea. Sequence analysis of the N terminus and the two heme-containing peptides generated by digestion of the enzyme with trypsin show 40% homology overall to sequences reported for the di-heme peroxidase from Pseudomonas aeruginosa (Rönnberg, M., Kalkkinen, N., and Ellfolk, N. (1989) FEBS Lett. 250, 175-178). At room temperature and pH 7.0, one heme is low spin with Em7 = +450 mV and the other is high spin with Em7 = -260 mV. EPR spectra show a mixture of high spin and low spin signals at cryogenic temperatures. Anionic ligands (CN-, N3-, F-, CNO-) bind so as to perturb the high spin heme when cytochrome c553 peroxidase is either fully oxidized (FeLS3+:FeHS3+) or half-reduced (FeLS2+:FeHS3+). The EPR signal of the high potential, low spin heme in fully oxidized enzyme is unperturbed by the presence of the ligands. Furthermore, each ligand results in similar characteristic EPR signals for either oxidation state of the peroxidase. Both the fully oxidized and half-reduced oxidation states of cytochrome c553 peroxidase are catalytically active as evidenced by the enzyme's ability to oxidize horse heart cytochrome c in the presence of H2O2, as well as by optical changes associated with the addition of H2O2 to the peroxidase. In the presence of stoichiometric amounts of H2O2, the half-reduced enzyme is rapidly oxidized and the fully oxidized enzyme shows a significant decrease in absorbance in the Soret region of the optical spectrum coupled with a lesser increase near 600-650 nm. These latter optical changes are similar to what is observed in the formation of a porphyrin cation radical. This suggests that this di-heme peroxidase may form a compound I intermediate analogous to that formed by horseradish peroxidase.

Amino Acid Sequence↗
PubMed1994

Identification of axial ligands of cytochrome c552 from Nitrosomonas europaea.

Cytochrome c552 from Nitrosomonas europaea was analyzed by visible, EPR and MCD spectroscopies. The visible and MCD data show that histidine and methionine are the axial ligands to the heme iron of the ferric protein. The EPR spectrum of the cytochrome shows an atypical highly axial low spin (HALS) type signal with g-values that make it difficult to identify the axial ligands. These results reinforce the value of near-infrared MCD spectroscopy for assigning ligands in ferric heme systems and point out the difficulties in using only EPR spectroscopy for the same purpose. The description of another c-cytochrome exhibiting a HALS-type EPR signal will eventually be helpful in explaining the physical basis for this unusual signal.

Circular Dichroism↗
PubMed1994

Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme c cytochromes.

The organization of genes for three proteins involved in ammonia oxidation in Nitrosomonas europaea has been investigated. The amino acid sequence of the N-terminal region and four heme-containing peptides produced by proteolysis of the tetraheme cytochrome c554 of N. europaea were determined by Edman degradation. The gene (cycA) encoding this cytochrome is present in three copies per genome (H. McTavish, F. LaQuier, D. Arciero, M. Logan, G. Mundfrom, J.A. Fuchs, and A. B. Hooper, J. Bacteriol. 175:2445-2447, 1993). Three clones, representing at least two copies of cycA, were isolated and sequenced by the dideoxy-chain termination procedure. In both copies, the sequences of 211 amino acids derived from the gene sequence are identical and include all amino acids predicted by the proteolytic peptides. In two copies, the cycA open reading frame (ORF) is followed closely (three bases in one copy) by a second ORF predicted to encode a 28-kDa tetraheme c cytochrome not previously characterized but similar to the nirT gene product of Pseudomonas stutzeri. In one copy of the cycA gene cluster, the second ORF is absent.

Amino Acid Sequence↗
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↗
PubMed1993

Hydroxylamine oxidoreductase from Nitrosomonas europaea is a multimer of an octa-heme subunit.

A fully active form of hydroxylamine oxidoreductase from Nitrosomonas has been purified with high recovery and shown by reverse-phase high performance liquid chromatography and N-terminal analysis to contain only a 63-kDa subunit and to lack the 11-kDa protein previously thought to be a second subunit. Based on the previously published values of molecular weight in solution, hydroxylamine oxidoreductase probably has an alpha 2 or alpha 3 oligomeric structure. The enzyme was digested separately with trypsin and chymotrypsin and peptides which contained covalently bound heme were separated by high performance liquid chromatography and their amino acid sequences determined. A total of seven heme-containing peptides of unique amino acid sequence were obtained. Six of these heme-containing peptides clearly contained a single c-heme with optical properties indistinguishable from the tryptic heme-containing peptide from horse heart cytochrome c. No noncovalently bound heme was observed. One of the seven heme-containing peptides (T7) was unusual in that it released 2 amino acid residues after each cycle of the Edman degradation due to a nondisulfide cross-link and exhibited a Soret band that was broadened in both the ferric form at neutral pH and the pyridine ferrohemochrome. Subdigestion of peptide T7 with nonspecific proteases (Pronase, bromelain, or pepsin) resulted in the isolation of two smaller heme-containing peptides of unique sequences. One of these was spectrally identical to the other c-heme containing peptides, whereas the second was still apparently cross-linked, again releasing 2 amino acid residues after each Edman cycle. This second peptide possessed a heme-like chromophore with absorption bands (Soret, alpha and beta) red-shifted about 6 nm relative to the spectrum of c-heme-containing peptides. Thus, hydroxylamine oxidoreductase contains a total of eight covalently bound hemes per subunit, seven of which are c-hemes. The eighth, which is attached to a cross-linked peptide, is probably the unusual P460 heme which is unique to hydroxylamine oxidoreductase and thought to be at the active site.

Amino Acid Sequence↗
PubMed1993

Optical spectropotentiometric resolution of the hemes of hydroxylamine oxidoreductase. Heme quantitation and pH dependence of Em.

The hemes of hydroxylamine oxidoreductase (HAO) have been analyzed optically by potentiometric titrations using a low volume optically transparent thin layer electrochemical cell. The electrochemical behavior of the HAO monomeric unit has been interpreted by modeling the spectroelectrochemical data at several wavelengths to eight one-electron Nernst sites: seven c-type hemes and one P460 heme. Of the seven c-hemes, six show alpha-bands with absorption maxima at or near 553 nm. One c-heme has an alpha-band absorption maximum at 559 nm. The six c-553 hemes have midpoint potentials at pH 7.0 of +288, -10, -162, -192, -265 and -412 mV versus the normal hydrogen electrode (NHE). The c-559 heme has a midpoint potential (Em') at pH 7.0 of +11 mV versus NHE. The midpoint potential of the P460 heme is at -260 mV versus NHE at pH 7.0. In contrast, the midpoint potential for the P460 heme in another protein, cytochrome P460, from the same organism is -402 mV versus NHE at pH 7.0. Midpoint potentials of the c-hemes show little, if any, pH dependence over the range of pH 6-8. In contrast, Em' for the P460 heme changes with a slope of -60 mV/pH unit over the same range. Electrochemical isolation of the P460 heme at pH 8.0 led to the discovery of a broad spectroscopic feature centered near 740 nm that was assigned to the oxidized P460 heme. Changes in the spectroelectrochemical behavior of HAO after inactivation by H2O2 was almost exclusively restricted to the P460 heme of HAO. Both the 464-nm absorption band of the reduced P460 heme and the 740-nm band of the oxidized heme were no longer present. For the c-hemes, the only effect seems to be a slight shift in Em' for a single c-553 heme from -162 to -135 mV.

Cytochromes↗
PubMed1993

Reductive dehalogenation of the trichloromethyl group of nitrapyrin by the ammonia-oxidizing bacterium Nitrosomonas europaea.

Suspensions of Nitrosomonas europaea catalyzed the reductive dehalogenation of the commercial nitrification inhibitor nitrapyrin (2-chloro-6-trichloromethylpyridine). The product of the reaction was identified as 2-chloro-6-dichloromethylpyridine by its mass fragmentation and nuclear magnetic resonance spectra. A small amount of 2-chloro-6-dichloromethylpyridine accumulated during the conversion of nitrapyrin to 6-chloropicolinic acid in an aerated solution in the presence of ammonia (T. Vannelli and A.B. Hooper, Appl. Environ. Microbiol. 58:2321-2325, 1992). Nearly stoichiometric conversion of nitrapyrin to 2-chloro-6-dichloromethylpyridine occurred at very low oxygen concentrations and in the presence of hydrazine as a source of electrons. Under these conditions the turnover rate was 0.37 nmol of nitrapyrin per min per mg of protein. Two specific inhibitors of ammonia oxidation, acetylene and allylthiourea, inhibited the rate of the dehalogenation reaction by 80 and 84%, respectively. In the presence of D2O, all 2-chloro-6-dichloromethylpyridine produced in the reaction was deuterated at the methyl position. In an oxygenated solution and in the presence of ammonia or hydrazine, cells did not catalyze the oxidation of exogenously added 2-chloro-6-dichloromethylpyridine to 6-chloropicolinic acid. Thus, 2-chloro-6-dichloromethylpyridine is apparently not an intermediate in the aerobic production of 6-chloropicolinic acid from nitrapyrin.

Ammonia↗
PubMed1993

Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.

Acetylene↗
PubMed1993

Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea.

The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.

Amino Acid Sequence↗
PubMed1992

Pressure modulation of cytochrome-to-cytochrome electron-transfer. Models and enzyme reactions.

The kinetics of electron-transfer involved in reactions of reduction of 2,6-dichlorophenol indophenol and Fe(CN)3-(6) by L-ascorbic acid and reduction of ferric cytochrome c by both L-ascorbic acid and reduced hydroxylamine oxidoreductase were studied as a function of three parameters: ionic strength, pressure (1-2000 bar) and temperature (4-20 degrees C) using the high-pressure stopped-flow method. From measurements, the thermodynamic parameters of activation volume (delta V++), and, when possible, activation enthalpy and entropy (delta H++ and delta S++) have been calculated. We found, for these four systems, that the pressure has revealed solvation effects involved in electron-transfer. For the reduction of ferric cytochrome c by reduced hydroxylamine oxidoreductase (a cytochrome-to-cytochrome electron-transfer), we have not obtained evidence for a conformational change.

2,6-Dichloroindophenol↗
PubMed1992

Oxidation of Nitrapyrin to 6-Chloropicolinic Acid by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea.

Suspensions of Nitrosomonas europaea catalyzed the oxidation of the commercial nitrification inhibitor nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine]. Rapid oxidation of nitrapyrin (at a concentration of 10 muM) required the concomitant oxidation of ammonia, hydroxylamine, or hydrazine. The turnover rate was highest in the presence of 10 mM ammonia (0.8 nmol of nitrapyrin per min/mg of protein). The product of the reaction was 6-chloropicolinic acid. By the use of O(2), it was shown that one of the oxygens in 6-chloropicolinic acid came from diatomic oxygen and that the other came from water. Approximately 13% of the radioactivity of [2,6-C]nitrapyrin was shown to bind to cells. Most (94%) of the latter was bound indiscriminately to membrane proteins. The nitrapyrin bound to membrane proteins may account for the observed inactivation of ammonia oxidation.

Journal Article↗
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↗