c-cytochromes of the ammonia-oxidizing chemolithoautotrophic bacteria.
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Ammonia↗
Biomedical subjects
A B Hooper
Publications and source records attributed to A B Hooper.
P460 of hydroxylamine oxidoreductase of Nitrosomonas europaea: Soret resonance Raman evidence for a novel heme-like structure.
P460, an iron-containing chromophore at the active site of Hydroxylamine Oxidoreductase of the ammonia-oxidizing bacterium Nitrosomonas europaea, is a macrocycle of unknown structure with a Soret-like 460-nm absorption band in the ferrous form. The pigment can also be isolated in a peptide, "P460-Fragment". Resonance Raman spectroscopy (lambda ex = 457.9 nm) suggests that P460 is a new type of heme with symmetry properties lower than those of protophorphyrin IX or chlorins and similar to those of chlorophylls and isobacteriochlorins. Some of the resonance Raman vibrations of P460 are shifted in HAO as compared to those of P460-Fragment.
Binding Sites↗
Electrophoresis at elevated hydrostatic pressure of the multiheme hydroxylamine oxidoreductase.
The behavior of the multiheme protein hydroxylamine oxidoreductase (HAO) in polyacrylamide gel electrophoresis was studied at hydrostatic pressures up to 3 kbar at 25 degrees C. Due to the limited working volume of the high pressure vessel, the electrophoresis cells were miniaturized. A microcell which accommodates 6 capillary gel tubes is described. Between 1 bar and 1.5 kbar the enzyme did not undergo structural changes detectable in the gel system. At approximately 2 kbar the active form of the enzyme was partially dissociated. At higher pressures, the enzyme was converted to forms which were irreversibly inactive and had a higher apparent molecular mass, suggesting aggregation or denaturation.
Atmospheric Pressure↗
Degradation of halogenated aliphatic compounds by the ammonia- oxidizing bacterium Nitrosomonas europaea.
Suspensions of Nitrosomonas europaea catalyzed the ammonia-stimulated aerobic transformation of the halogenated aliphatic compounds dichloromethane, dibromomethane, trichloromethane (chloroform), bromoethane, 1,2-dibromoethane (ethylene dibromide), 1,1,2-trichloroethane, 1,1,1-trichloroethane, monochloroethylene (vinyl chloride), gem-dichloroethylene, cis- and trans-dichloroethylene, cis-dibromoethylene, trichloroethylene, and 1,2,3-trichloropropane, Tetrachloromethane (carbon tetrachloride), tetrachloroethylene (perchloroethylene), and trans-dibromoethylene were not degraded.
Ammonia↗
Degradation of trichloroethylene by the ammonia-oxidizing bacterium Nitrosomonas europaea.
Suspensions of Nitrosomonas europaea are shown to cause the complete disappearance of 10 microM trichloroethylene at rates of 1 microM mg protein-1. The reaction continues at nearly this rate for many hours. Fresh cells catalyze the reaction in the absence of added ammonium (presumably utilizing endogenous ammonia or stored reductant). In older cells, trichloroethylene degradation depends on the addition of ammonia. Acetylene, 2-chloro 6-trichloromethylpyridine and alpha alpha'dipyridyl, which inhibit the oxidation of ammonia by cells, inhibit the degradation of trichloroethylene. Thus degradation of trichloroethylene is dependent on- and possibly catalyzed by the ammonia oxidizing enzyme.
Ammonia↗
Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase. Evidence for conformational change.
Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)
Carbon Monoxide↗
Conformational change accompanies redox reactions of the tetraheme cytochrome c-554 of Nitrosomonas europaea.
Cytochrome c-554 of the ammonia-oxidizing chemolithoautotropic bacteria is thought to mediate electron transfer from hydroxylamine oxidoreductase to a terminal oxidase and/or to ammonia monooxygenase. The cytochrome has four c hemes which interact magnetically and have the same redox potential. We report that the kinetics of reduction of ferric cytochrome c-554 by dithionite or the oxidation of ferrous cytochrome c-554 by O2 or H2O2 are complex and multiphasic. Transient rapid-scan difference spectra indicate discrete maxima at approximately 418 nm, 425 nm and 432 nm. Absorbance changes at all three difference maxima appear to occur in all kinetic phases, although not in equal amounts for each wavelength. Reduction by 20 mM dithionite was biphasic. At pH 7.5 the first phase, which involved approximately 50% of the total absorbance change, had a rate constant (20 degrees C) of 140 s-1 and energy of activation of 20 kJ X mol-1. The slow phase had a rate constant 0.43 s-1 and a relatively high energy of activation, 87 kJ X mol-1, suggesting that a change in protein configuration accompanied the reaction. As the pH of the solution increased, the rate constant for both phases decreased and the fraction of absorbance change in the rapid phase increased. Oxidation of ferrous cytochrome c-554 by O2 involved a discrete rapid phase with a rate constant of 14 s-1, accounting for 6% of the absorbance. The remainder of the reaction was multiphasic with rate constants in the range 0.1-0.01 s-1. With H2O2 as the oxidant, the rapid phase involved 39% of the change in absorbance with a rate constant of 19 s-1. The remainder of the reoxidation was multiphasic with rate constants ranging over 0.4-0.01 s-1.
Cytochrome c Group↗
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.
Amino Acids↗
Oxygen exchange between nitrate molecules during nitrite oxidation by Nitrobacter.
During oxidation of nitrite, cells of Nitrobacter winogradskyi are shown to catalyze the active exchange of oxygen atoms between exogenous nitrate molecules (production of 15N16/18O3- during incubation of 14N16/18O3-, 15N16O3-, and 15N16O2- in H216O). Little, if any, exchange of oxygens between nitrate and water also occurs (production of 15N16/18O3- during incubation of 15N16O3- and 14N16O2- in H218O). 15N species of nitrate were assayed by 18O-isotope shift in 15N NMR. Taking into account the O-exchange reactions which occur during nitrite oxidation, H2O is seen to be the source of O in nitrate produced by oxidation of nitrite by N. winogradskyi. The data do not establish whether the nitrate-nitrate O exchange is catalyzed by nitrite oxidase (H2O + HNO2----HNO3 + 2H+ + 2e-) or nitrate reductase (HNO3 + 2H+ + 2e-----HNO2 + H2O) or both enzymes in consort. The nitrate-nitrate exchange reaction suggests the existence of an oxygen derivative of a H2O-utilizing oxidoreductase.
Kinetics↗
Tetraheme cytochrome c-554 from Nitrosomonas europaea. Heme-heme interactions and ligand binding.
Cytochrome c-554 functions in the ammonia oxidizing system of Nitrosomonas europaea. We have investigated its molecular and ligand binding properties and studied the protein with optical, EPR, and Mössbauer spectroscopies in the pH range from 2 to 13. Amino acid, heme, and metal analyses show that the protein has Mr = 25,000 and that it contains four c-type hemes per molecule. Optical spectra reveal that the heme ligand structures are sensitive to the pH of the medium and that the hemes can bind small molecules such as CN-, CO, and NO under certain conditions. According to the Mössbauer and EPR studies of the ferric protein, the hemes are predominantly (75%) high spin at pH 2 and low spin (approximately equal to 100%) above pH 10. At neutral pH, Mössbauer data show that 75% of the heme is low spin and that the remainder is high spin. The EPR data, however, do not reveal any signals attributable to typical high spin or low spin species. Rather, a very complex and unusual spectrum with a main feature at g = 3.3 is observed at X-band, this feature shifts to approximately g = 3 at S-band. The EPR and Mössbauer data show clearly that the hemes are magnetically interacting, by dipolar and exchange interactions. At pH 2, the EPR spectra reveal resonances at g = 6 and 2. The Mössbauer spectra prove that all hemes are magnetically coupled at this pH. Coupling is also borne out by the observation of a half-field EPR resonance near g = 12.
Amino Acids↗
In bacteria which grow on simple reductants, generation of a proton gradient involves extracytoplasmic oxidation of substrate.
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Ammonia↗
Kinetics of reduction by substrate or dithionite and heme-heme electron transfer in the multiheme hydroxylamine oxidoreductase.
Hydroxylamine oxidoreductase of Nitrosomonas catalyzes the dehydrogenation of NH2OH. It contains hemes c553, c559 and P460 in the ratio 5:2:1. At equilibrium four or five c hemes are reduced by NH2OH or NH2NH2, respectively. Heme P460 is the site of electron entry into the enzyme; electrons exit via P460 to O2 or H2O2 with rate constants of 30s-1. We report that hydroxylamine oxidoreductase has two categories of electron-accepting sites: (a) heme P460, an H2O2-sensitive site, which is reactive with NH2OH (2.2 hemes c557 and 2 hemes c559 are reduced) or NH2NH2 (3.3 heme c 553 and 2 heme c559 are reduced) and (b) an H2O2-insensitive site(s) which is reactive with H2O2 (approximately 0.15 heme c553 is reduced); hydroquinone, pyrogallol, N-methyl hydroxylamine, pyocyanine, and ascorbate (approximately 0.8 heme c553 is reduced); or Na2S2O4 or EDTA-photoreduction with proflavin, deazalumiflavin or acridine orange and methylviologen (all hemes are reduced). The rate constants at 19 degrees C for reduction by dithionite were: 0.7 heme c553 (7s-1), 4.3 hemes c553 (0.07 s-1), 0.7 heme c559 (0.8s-1), 1.3 hemes c559 (0.1s-1), P460 (0.013s-1). At 2 degrees C the rate constant for 0.8 heme c559 was 1.7s-1. The data indicate that one heme c552 is reduced by dithionite at the same rate as mammalian cytochrome c; other hemes are reduced much more slowly and are possibly inaccessible to the solvent. The rate constants at 2 degrees C for reduction by NH2OH were: 1.8 hemes c553 (30s-1), 0.2 heme c553 (2.4s-1), 1.7 hemes c559 (19s-1), 0.3 heme c559 (1.4s-1). For reduction by NH2NH2 the values were: 2.6 hemes c553 (23s-1), 0.7 heme c553 (1.6s-1), 1.3 hemes c559 (22s-1), 0.7 heme c559 (4.2s-1). Thus reduction by NH2OH at the substrate site was at least an order of magnitude faster than reduction of hydroxylamine oxidoreductase heme by Na2S2O4. Comparison of rates of heme-heme electron transfer on the enzyme during reoxidation by O2 or H2O2, reduction by Na2S2O4 and reduction by NH2OH or NH2NH2 indicates that the enzyme can exist in distinct states which result in different rates of heme-heme electron transfer. Comparison of the rate of substrate reduction of c hemes of hydroxylamine oxidoreductase (HAO) with the turnover of the enzyme in vivo is consistent with the electron path NH2OH----HAO P460----HAO c hemes----biological electron acceptor.
Anaerobiosis↗
Mössbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas. A ferrous heme with an unusually large quadrupole splitting.
Hydroxylamine oxidoreductase from Nitrosomonas europeae catalyzes the oxidative conversion of NH2OH to NO-2. The enzyme, Mr = 220,000, has an (alpha beta)3 subunit structure with each alpha beta subunit containing 7-8 c-type hemes and one unusual prosthetic group, termed P-460. The P-460 is also found in a Mr approximately equal to 17,000 protein (P-460 fragment). Mössbauer spectra of the reduced P-460 groups, in hydroxylamine oxidoreductase and the fragment, exhibit nearly identical quadrupole doublets with an unusually large splitting, delta EQ = 4.21 mm/s (no ferrous heme protein is known with delta EQ greater than 2.75 mm/s). The observed isomer shift, delta = 0.96 mm/s at 4.2 K, shows that the P-460 iron is high spin ferrous. Treatment of oxidized hydroxylamine oxidoreductase with H2O2 followed by reduction or exposure of the native sample to CO led to the disappearance of both the characteristic 460 nm absorption band (epsilon = 89 mM-1 cm-1) and the delta EQ = 4.21 mm/s doublet. The iron of the oxidized P-460 fragment is high spin ferric, with Mössbauer and EPR parameters very similar to those of metmyoglobin. Optical spectra of the reduced P-460 fragment show long wavelength bands at 650 and 688 nm which are sensitive to treatment of the fragment with reagents which react with P-460. These bands were, however, not detected in hydroxylamine oxidoreductase. The spectroscopic and chemical evidence obtained to date suggests strongly that the P-460 iron resides in a heme-like macrocycle although the presumed porphyrin must have some unusual features.
Electron Spin Resonance Spectroscopy↗
Diheme cytochrome c-554 from Nitrosomonas. Soret resonance Raman indication of an unusual ferric 5-coordinate structure.
The diheme cytochrome c-554 which participates in ammonia oxidation in the chemoautotroph , Nitrosomonas europaea has been studied by Soret excitation resonance Raman spectroscopy. The Raman spectrum of reduced cytochrome c-554 at neutral pH is similar classical 6-coordinate low-spin ferrous mammalian cytochrome c. In contrast, the spectrum of ferric cytochrome c-554 suggests a 5-coordinate state which is unusual for c hemes. The oxidized spectrum closely resemble that of horseradish peroxidase (HRP) or cytochrome c peroxidase (CcP) at pH 6.4. The narrow linewidth of the heme core-size vibrations indicates that both heme irons of c-554 have similar geometries.
Cytochrome c Group↗
Resolution of the hemes of hydroxylamine oxidoreductase by redox potentiometry and optical spectroscopy.
Optical spectroscopy combined with redox potentiometry has resolved the hemes of hydroxylamine oxidoreductase into 6 thermodynamically distinct classes. There are apparently 4 classes of heme c553, with Em7-values of 295 mV, 10 mV, -190 mV and -390 mV, present in a stoichiometry of 1:1:2:1; two equivalents of heme c559, Em7 O mV, and one of heme P-460, an unusual chromophore, with Em7 -260 mV.
Heme↗
Heme P460 of hydroxylamine oxidoreductase of Nitrosomonas. Reaction with CO and H2O2.
Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO-binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2-oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate-limiting. Activation of dioxygen for N-oxidation by ferrous HAO may involve the two-electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen. Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 X 10(-3) M-1 s-1 and Kd, 12 microM.
Carbon Monoxide↗
Evaluating pedigree data. II. Identifying the cause of error in families with inconsistencies.
Pedigree data can be evaluated, and subsequently corrected, by analysis of the distribution of genetic markers, taking account of the possibility of mistyping . Using a model of pedigree error developed previously, we obtained the maximum likelihood estimates of error parameters in pedigree data from Tokelau. Posterior probabilities for the possible true relationships in each family are conditional on the putative relationships and the marker data are calculated using the parameter estimates. These probabilities are used as a basis for discriminating between pedigree error and genetic marker errors in families where inconsistencies have been observed. When applied to the Tokelau data and compared with the results of retyping inconsistent families, these statistical procedures are able to discriminate between pedigree and marker error, with approximately 90% accuracy, for families with two or more offspring. The large proportion of inconsistencies inferred to be due to marker error (61%) indicates the importance of discriminating between error sources when judging the reliability of putative relationship data. Application of our model of pedigree error has proved to be an efficient way of determining and subsequently correcting sources of error in extensive pedigree data collected in large surveys.
Female↗
Evaluating pedigree data. I. The estimation of pedigree error in the presence of marker mistyping.
Pedigrees used in the analysis of genetic or medical data are usually ascertained from sources subject to a variety of errors including misidentification of individuals, faults in historical documents or record linkage, nonpaternity, and unidentified adoption. Genetic markers can be used to verify putative family and pedigree data through the search for inconsistencies, or genetic exclusions, between putative parents and offspring. The probability of observing an exclusion given the occurrence of an error depends upon the gene frequencies at the loci under study and the forms of error. In addition, inconsistencies can arise from laboratory errors in marker determination. Together, these problems make the proper statistical analysis of such data desirable. Here we give a model that specifies the combined effects of various kinds of pedigree error along with genetic marker error. This model allows the maximum-likelihood estimation of the rates of various forms of pedigree error and laboratory error from genetic marker data collected on putative families. The method is illustrated by applying it to data obtained from a South Pacific island population, Tokelau. From the observed distribution of genetic marker inconsistencies between the parents and offspring of putative families, derived from the extensive genealogy of this population, we are able to estimate that the error of a paternal link is 4%, the error of a maternal link is zero, and the overall system typing error is 1%.
Alleles↗