Antimicrob Agents Chemother, 1988 Feb, 32(2), 276 - 8 In vitro susceptibility of Alcaligenes denitrificans subsp . xylosoxidans to 24 antimicrobial agents; Glupczynski Y et al.; The in vitro susceptibilities of 37 clinical isolates of Alcaligenes denitrificans subsp . xylosoxidans to 24 antimicrobial agents were determined . Imipenem was the only drug with consistent activity (MIC for 90% of isolates, 2 micrograms/ml) . Piperacillin, ticarcillin-clavulanic acid, ceftazidime, and co-trimoxazole were active against most strains . All the isolates were resistant to ampicillin, cefazolin, cefuroxime, cefamandole, cefotetan, ceftriaxone, cefotaxime, aztreonam, amdinocillin, and temocillin . Most isolates were resistant to the aminoglycosides tested, including amikacin . Lack of activity was also observed for all new 4-quinolone antimicrobial agents. Biochim Biophys Acta, 1988 Jan 12, 964(1), 83 - 9 Examination of differences between benzo{a}pyrene and steroid hydroxylases in guinea pig adrenal microsomes; Mochizuki H et al.; The effects of antibodies to cytochromes P-45017 alpha,lyase and P-450C21 on benzo{a}pyrene hydroxylase activity were measured in microsomes from guinea pig adrenals . Anti-cytochrome P-45017 alpha,lyase IgG inhibited about 30% of the benzo{a}pyrene hydroxylase activity of the microsomes in the presence of excess amounts of the IgG, but anti-cytochrome P-450C21 IgG did not affect the activity . In a reconstituted system, consisting of cytochrome P-450, NADPH-cytochrome-P-450 reductase and dilauroylphosphatidylcholine, cytochrome P-45017 alpha,lyase catalyzed, in addition to steroid hydroxylation, benzo{a}pyrene hydroxylation, but cytochrome P-450C21 did not hydroxylate benzo{a}pyrene . Since anti-cytochrome P-45017 alpha,lyase IgG inhibited benzo{a}pyrene hydroxylase activity completely in the reconstituted system with cytochrome P-45017 alpha,lyase, the presence of non-inhibited benzo{a}pyrene hydroxylase in the microsomes suggests that the residual activity in the microsomes may be due to some enzyme other than cytochrome P-45017 alpha,lyase . Benzo{a}pyrene hydroxylase activity was detected in detergent-solubilized microsomes from which cytochrome P-45017 alpha,lyase was removed by using an immobilized anti-cytochrome P-45017 alpha,lyase IgG-Sepharose column . This shows the existence in microsomes of a benzo{a}pyrene hydroxylase other than cytochrome P-45017 alpha,lyase . Benzo{a}pyrene hydroxylase in the solubilized microsomes required O2, NADPH and NADPH-cytochrome-P-450 reductase for its activity and was inhibited by CO . This suggests that benzo{a}pyrene hydroxylase is a cytochrome P-450-dependent monooxygenase . This novel enzyme was also active in xenobiotic metabolism, such as 2-nitropropane denitrification and aminopyrine demethylation. Appl Environ Microbiol, 1988 Jan, 54(1), 74 - 8 Environmental factors affecting indole metabolism under anaerobic conditions; Madsen EL et al.; The influence of physiological and environmental factors on the accumulation of oxindole during anaerobic indole metabolism was investigated by high-performance liquid chromatography . Under methanogenic conditions, indole was temporarily converted to oxindole in stoichiometric amounts in media inoculated with three freshwater sediments and an organic soil . In media inoculated with methanogenic sewage sludge, the modest amounts of oxindole detected at 35 degrees C reached higher concentrations and persisted longer when the incubation temperature was decreased from 35 to 15 degrees C . Also, decreasing the concentration of sewage sludge used as an inoculum from 50 to 1% caused an increase in the accumulation of oxindole from 10 to 75% of the indole added . Under denitrifying conditions, regardless of the concentration or source of the inoculum, oxindole appeared in trace amounts but did not accumulate during indole metabolism . In addition, denitrifying consortia which previously metabolized indole degraded oxindole with no lag period . Our data suggest that oxindole accumulation under methanogenic, but not under denitrifying conditions is caused by differences between relative rates of oxindole production and destruction. Arch Microbiol, 1988, 150(4), 358 - 62 Anaerobic metabolism of cyclohexanol by denitrifying bacteria; Dangel W et al.; Three strains of denitrifying bacteria were anaerobically enriched and isolated from oxic or anoxic habitats with cyclohexanol or cyclohexanone as sole electron donor and carbon source and with nitrate as electron acceptor . The bacteria were facultatively anaerobic, Gram-negative and metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as terminal electron acceptor . Cyclohexanol and cyclohexanone were degraded both anaerobically and aerobically . Aromatic compounds were oxidized in the presence of molecular oxygen only . One of the bacterial strains was further characterized . During anaerobic cyclohexanol degradation approximately 40% of the substrate was oxidized to phenol, which accumulated as dead-endproduct in the growth medium; 60% of cyclohexanol was completely oxidized to CO2 and assimilated, respectively . In addition to phenol formation, transient accumulation of cyclohexanone, 2-cyclohexenone and 1,3-cyclohexanedione was observed . Based on these findings we propose a pathway for anaerobic cyclohexanol degradation involving these intermediates. J Gen Microbiol, 1988 Jan, 134 ( Pt 1), 221 - 6 Nitrosamine formation by denitrifying and non-denitrifying bacteria: implication of nitrite reductase and nitrate reductase in nitrosation catalysis; Calmels S et al.; Biochemical, microbiological and genetic studies were done to characterize the mechanism of bacterial formation of N-nitrosomorpholine (NMOR) from morpholine and nitrite at neutral pH . In Escherichia coli and Proteus morganii, the nitrosating activity was markedly induced when bacteria were cultured under anaerobiosis in minimal medium containing nitrate, while in the presence of nitrite there was no induction . However, induction of the nitrosating activity in Pseudomonas aeruginosa occurred in anaerobic cultures in the presence of either nitrate or nitrite . The nitrosation capacity was also examined in various E . coli K12 mutants whose structural gene of either nitrate reductase or nitrite reductase was deleted . Nitrosation was not linked to the three (NADH-, formate- and glucose-dependent) nitrite reductases but was directly dependent on the presence of a nitrate reductase. Prog Clin Biol Res, 1988, 274, 653 - 68 Evolution of cytochrome c oxidase; Kadenbach B et al.; The current view on the regulatory function of nuclear-encoded subunits of cytochrome c oxidase from higher evolved organisms is presented . The activity of monomeric laurylmaltoside-dissolved, but not of reconstituted cytochrome c oxidase, is strongly affected by anions accompanied by a conformational change of the enzyme, as shown by changed visible spectra . Addition of uncoupler to proteoliposomes induces the same anion sensitivity as obtained with the soluble enzyme, suggesting dissociation of the dimeric membrane-bound enzyme by uncoupler . Nucleotides are suggested to regulate cytochrome c oxidase activity at 3 different sites: 1) Interaction of ATP with a cytosolic site (outside) increases the Km for cytochrome c in the enzyme from bovine heart and Paracoccus denitrificans; 2) binding of ADP at a matrix site decreases, and 3) binding of ATP at another matrix site increases the Km for cytochrome c of the mammalian enzyme. Prog Clin Biol Res, 1988, 274, 619 - 35 Kinetics of the interaction of cytochrome c oxidase of Paracoccus denitrificans with Paracoccus and mitochondrial cytochrome c; Smith L et al.; We have studied the reactions of the oxidase of Paracoccus dentrificans with its membrane-bound cytochrome c and with soluble cytochrome c550 of Paracoccus and of bovine heart . The turnover rate of Paracoccus oxidase with membrane-bound cytochrome c is high, approaching 1000/sec . at 25 degrees . When soluble cytochrome c is added to the electron transport chain oxidizing NADH or succinate, no increase in 02 uptake is observed . When the oxidase is reacting with the membrane-bound cytochrome c, the reaction site is not exposed for reaction with soluble cytochrome c . We have purified the Paracoccus oxidase, following relatively simple methodology . It has three major subunits similar in molecular weight to those of the larger subunits of the bovine oxidase . When reconstituted in the presence of asolectin, it is just as active as the intact membrane-bound oxidase in reaction with soluble cytochrome c . The soluble cytochrome c reacts directly with the cytochrome aa3 . We found direct evidence that the oxidase is stimulated in the presence of low concentrations of cytochrome c . The stimulatory effect could be the explanation for the so-called "high affinity" site for reaction with cytochrome c . The reaction of bovine cytochrome c with Paracoccus oxidase resembles that with the bovine oxidase in every way tested . The Paracoccus oxidase must have a cytochrome c binding site equivalent to that of the bovine enzyme . The reaction of the Paracoccus oxidase with its own soluble cytochrome c550, which has a highly negative hemisphere on the side of the molecule away from the heme crevice, has different properties from those seen in its reaction with bovine cytochrome c . However the properties all change to be like those with bovine cytochrome c on addition of poly-L-lysine . These data emphasize the importance of all of the charged groups on the cytochrome c in influencing binding or electron transfer reactions . The respiratory chain on the membranes of a cytochrome c-deficient mutant can reduce cytochrome aa3 using NADH as substrate in a manner similar to that of the wild type, although at somewhat lower rate, suggesting diffusional encounter of the large complexes within the membrane . Our data permit speculations about the possible evolution from the bacterial to the mitochondrial electron transport system. Infection, 1988, 16 Suppl 2, S89 - 91 Taxonomy of the genus Listeria; Rocourt J; According to DNA homology values as well as to 16S rRNA cataloguing results, the genus Listeria presently contains two groups of closely related species: one is constitued by Listeria gravi and Listeria murrayi, the other by Listeria monocytogenes, Listeria ivanovii, Listeria innocua, Listeria welshimeri, and Listeria seeligeri . Among these seven species, only two are pathogenic for humans and animals: Listeria monocytogenes and Listeria ivanovii . Listeria denitrificans was recently excluded from this genus and transferred to a new genus, Jonesia, as Jonesia denitrificans. Appl Environ Microbiol, 1988 Jan, 54(1), 143 - 9 Biodegradation of alpha- and beta-hexachlorocyclohexane in a soil slurry under different redox conditions; Bachmann A et al.; Aerobic conditions proved to be best for the microbiol conversion of alpha-hexachlorocyclohexane (alpha-HCH) in a soil slurry . The dry soil contained 400 mg of alpha-HCH per kg . This xenobiotic compound was mineralized within about 18 days at an initial rate of 23 mg/kg of soil per day by the mixed native microbial population of the soil . The only intermediate that was detected during breakdown was pentachlorocyclohexene, which was detected at very small concentrations . Alpha-HCH was also bioconverted under methanogenic conditions . However, a rather long acclimation period (about 30 days) was necessary before degradation started, at a rate of 13 mg/kg of soil per day . Mass balance calculations showed that about 85% of the initial alpha-HCH that was present was converted to monochlorobenzene, 3,5-dichlorophenol, and a trichlorophenol isomer, possibly 2,4,5-trichlorophenol . Under both denitrifying and sulfate-reducing conditions, no significant bioconversion of alpha-HCH was observed . The beta isomer of HCH was recalcitrant at all of the four redox conditions studied . We propose that the specific spatial chloride arrangement of the beta isomer is responsible for its stability . The results reported here with complex soil slurry systems showed that alpha-HCH is, in contrast to the existing data in the literature, best degraded biologically in the presence of oxygen. Biochemistry, 1987 Dec 15, 26(25), 8059 - 65 Hydrogen bonding of sulfur ligands in blue copper and iron-sulfur proteins: detection by resonance Raman spectroscopy; Mino Y et al.; The resonance Raman spectrum of the blue copper protein azurin from Alcaligenes denitrificans exhibits nine vibrational modes between 330 and 460 cm-1, seven of which shift 0.4-3.0 cm-1 to lower energy after incubation of the protein in D2O . These deuterium-dependent shifts have been previously ascribed to exchangeable protons on imidazole ligands {Nestor, L., Larrabee, J . A., Woolery, G., Reinhammar, B., & Spiro, T . G . (1984) Biochemistry 23, 1084} or to exchangeable protons on amide groups which are hydrogen bonded to the cysteine thiolate ligands (a feature common to all blue copper proteins of known structure) . In order to distinguish between these two possibilities, a systematic investigation of Fe2S2(Cys)4-containing proteins was undertaken . Extensive hydrogen bonding between sulfur ligands and the polypeptide backbone had been observed in the crystal structure of ferredoxin from Spirulina platensis . The resonance Raman spectrum of this protein is typical of a chloroplast-type ferredoxin and exhibits deuterium-dependent shifts of -0.3 to -0.5 cm-1 in the Fe-S modes at 283, 367, and 394 cm-1 (assigned to the bridging sulfurs) and -0.6 to -0.8 cm-1 in the Fe-S modes at 328 and 341 cm-1 (assigned to the terminal cysteine thiolates) . Considerably greater deuterium sensitivity is observed in the Raman spectra of spinach ferredoxin and bovine adrenodoxin, particularly for the symmetric stretching vibration of the Fe2S2 moiety at approximately 390 cm-1 . This feature decreases by 0.8 and 1.1 cm-1, respectively, for the two oxidized proteins in D2O and by 1.8 cm-1 for reduced adrenodoxin in D2O.(ABSTRACT TRUNCATED AT 250 WORDS) Carcinogenesis, 1987 Dec, 8(12), 1907 - 12 Bacterially catalysed N-nitrosation reactions and their relative importance in the human stomach; Leach SA et al.; Human exposure to endogenously formed N-nitroso compounds has frequently been suggested as a causative factor in carcinogenesis where this is related to chronic bacterial infection such as is seen in gastric achlorhydria . At least two distinct mechanisms of endogenous formation have been identified . The first, a direct chemical reaction between secondary amino compounds and nitrite, is strongly pH dependent and does not proceed rapidly at neutral pH even in the presence of chemical catalysts . The second depends on the direct bacterial catalysis of N-nitrosation . The data presented demonstrate that the bacterially mediated reaction is catalysed by bacterial enzyme systems and proceeds much more rapidly at neutral pH than the chemical reaction . This suggests a particular relevance to the in vivo situation where neutral pH, bacteria and elevated nitrite concentrations are found . Drawing on the kinetic information presented regarding the bacterially mediated nitrosation reaction, the known kinetics of the chemical reaction and the published values for the relevant substrate concentrations in both the colonised and the normal acid stomach the bacterial and chemical reactions have been compared . Using these criteria, and assuming the presence of bacteria with the appropriate metabolic activity, it may be predicted that N-nitroso compounds may be formed in the colonized stomach at much higher concentrations than in the normal acid stomach . The difference in yield may be by two to four orders of magnitude . Different bacterial species and different isolates of the same species show considerable variation in their abilities to catalyse N-nitrosation reactions . The most rapid catalysis is associated with those bacteria capable of reducing nitrate and nitrite by the process of denitrification . The most significant clinical corollary of these studies is that although bacterial catalysis of N-nitrosation has been demonstrated unequivocally, bacterial colonization of the stomach may not itself necessarily result in elevated endogenous N-nitroso compound exposure despite the elevated nitrite concentrations normally associated with such colonization . An increase in exposure to endogenously formed N-nitroso compounds would only be predicted in those individuals where a significant proportion of the colonizing bacteria expressed significant N-nitrosation activity . As a consequence the carcinogenic risk may be restricted to only a small proportion of colonized individuals depending on the prevalence of sustained infection by bacteria with significant N-nitrosation activity, particularly denitrifiers. Biochem J, 1987 Dec 1, 248(2), 365 - 71 Amino acid sequences of cytochrome c-554(548) and cytochrome c' from a halophilic denitrifying bacterium of the genus Paracoccus; Ambler RP et al.; The amino acid sequences of the cytochromes c-554(548) and c' from the moderately halophilic bacterium Paracoccus sp., I.A.M . 203 (= A.T.C.C . 12084, N.C.I.B . 8669) have been determined . Cytochrome c-554(548) consists of a single polypeptide chain of 83 residues, and dimerizes strongly . The most similar protein of known sequence is the N-terminal half of the dihaem cytochrome c4, and other related proteins include the cytochrome c-554(547) of Thiobacillus neapolitanus and the cytochrome c-553 of Desulfovibrio vulgaris . Cytochrome c', which has a single polypeptide chain of 132 residues, is similar in sequence to cytochromes c' from phototrophic and denitrifying bacteria, but only shows about 36% sequence identity to the most similar protein of known sequence . Both of the Paracoccus proteins have a considerable excess of acidic amino acid side chains over basic ones, and a higher proportion of their basic amino acids is arginine than is usual in cytochromes c . Both these characteristics seem to be adaptations to increase the stability of the proteins in an environment of high ionic strength . Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication 50140 (24 pp.) at the British Library (Lending Division), Boston Spa, Yorkshire LS23 7BQ, U.K . from which copies are available on prepayment. Eur J Biochem, 1987 Dec 1, 169(2), 253 - 8 Optical, EPR and Mössbauer spectroscopic studies on the NO derivatives of cytochrome cd1 from Thiobacillus denitrificans; Liu MC et al.; We have used optical, EPR and Mossbauer spectroscopies to study the formation of heme-NO complex upon the addition of nitrite to reduced cytochrome cd1 from Thiobacillus denitrificans . The reduced d1 heme binds NO under both alkaline and acidic conditions, but the binding of NO to the reduced c heme was strongly pH-dependent . The Mossbauer data showed unambiguously that at pH 7.6 the c heme does not complex NO, whereas at pH 5.8 approximately half of the reduced c heme binds NO . This observation was confirmed by EPR studies, which showed that the spin concentration of the heme-NO EPR signal increased from 2 spins/molecule at pH 8.0 to approximately 3 spins/molecule at pH 5.8 . Optical absorption study also showed strong pH dependence in the binding of NO to the reduced c heme . We have also analyzed the Mossbauer spectra of the ferrous d1 heme-NO complex using a spin-Hamiltonian formalism . The magnetic hyperfine coupling tensor was found to be consistent with the unpaired electron residing on a sigma orbital. Biochem Biophys Res Commun, 1987 Nov 30, 149(1), 102 - 11 Immunological and spectral characterization of partly purified cytochrome oxidase from the cyanobacterium Synechocystis 6714; Wastyn M et al.; Membranes were isolated by French pressure cell extrusion of lysozyme-preincubated cells of the cyanobacterium Synechocystis 6714 after growth in the presence of 0.4 M NaCl for 4 days . These cells showed up to 6-fold respiratory activity (oxygen uptake) when compared to control cells . Separation of plasma and thylakoid membranes revealed that the major part of cytochrome c oxidase was associated with the latter . Immunoblotting of sodium dodecylsulfate polyacrylamide gel electrophorized membranes with antisera raised against subunit I, subunit II, and the holoenzyme of the aa3-type cytochrome oxidase from Paracoccus denitrificans gave specific and complementary cross-reactions at apparent molecular weights of about 25 and 17-18 kDa, respectively . Crude membranes were solubilized also with n-octyl glucoside, and the cytochrome oxidase was separated from the extract by affinity chromatography using immobilized cytochrome c from Saccharomyces cerevisiae . The enzyme was eluted with KCl/octyl glucoside . Dialysed and concentrated enzyme solution, which was free of b- and c-type cytochromes, gave reduced alpha- and gamma-peaks around 603 and 443 nm, respectively . Upon treatment of the sample with carbon monoxide the peaks were found at 593 and 433 nm, respectively . Photodissociation spectra of the CO-complexed enzyme were in full agreement with cytochrome aa3 being a functional cytochrome oxidase in Synechocystis 6714. J Infect, 1987 Nov, 15(3), 225 - 8 Septicaemia with probable endocarditis caused by Kingella denitrificans; Brown AM et al.; A 59-year-old man developed septicaemia caused by Kingella denitrificans . Treatment was with ampicillin and an aminoglycoside . The illness was complicated by a cerebral embolus . An obvious source of infection was not found. Microbiol Sci, 1987 Nov, 4(11), 329 - 33 The respiratory chain and energy conservation in the mitochondrion-like bacterium Paracoccus denitrificans; van Verseveld HW et al.; Paracoccus denitrificans has a flexible electron transport chain containing various inducible dehydrogenases, oxidases and reductases . The sequence of electron carriers and stoichiometry of energy transduction is well known, with the exception of all functional aspects of the different c-type cytochromes . The mitochondrion-like characteristics of the respiratory chain have stimulated much research at the molecular level, which should help to elucidate the structure-function relationship of the components of the different respiratory complexes isolated. J Bacteriol, 1987 Nov, 169(11), 4878 - 83 Plasmid pCBI carries genes for anaerobic benzoate catabolism in Alcaligenes xylosoxidans subsp . denitrificans PN-1; Blake CK et al.; Pseudomonas sp . strain PN-1 is reclassified as Alcaligenes xylosoxidans subsp . denitrificans PN-1 . Strain PN-1 is a gram-negative, rod-shaped organism, is motile by means of lateral flagella, is oxidase positive, and does not ferment sugars . Plasmid pCBI, carrying genes for the anaerobic degradation of benzoate in strain PN-1, is 17.4 kilobase pairs in length and is transmissible to a number of denitrifying Pseudomonas aeruginosa and Pseudomonas stutzeri strains . A restriction endonuclease map was constructed. Eur J Biochem, 1987 Oct 15, 168(2), 301 - 7 Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens; Zumft WG et al.; Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O . The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein . The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy . The protein was dimeric, with subunits of identical size (40 +/- 3 kDa) . Its pI was 6.05 . The EPR spectrum showed an axial signal g at 2.21(8) and g at 2.04(5) . The magnitude of the hyperfine splitting (A parallel = 6.36 mT) indicated the presence of type 1 copper only . The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (epsilon = 7.0 mM-1 cm-1), and a broad shoulder around 780 nm . A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P . aureofaciens . The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (epsilon = 2.5 mM-1 cm-1) and pronounced structures in the ultraviolet region . The EPR parameters were g parallel = 2.26(6) and g perpendicular = 2.05(6), with A parallel = 5.8 mT . The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide . The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 mumol substrate min-1 mg protein-1 . The reaction product again was nitric oxide . Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite . No 'oxidase' activity could be demonstrated for the enzyme . Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomonas. J Biol Chem, 1987 Oct 5, 262(28), 13805 - 11 The genes of the Paracoccus denitrificans bc1 complex . Nucleotide sequence and homologies between bacterial and mitochondrial subunits; Kurowski B et al.; The genes for the three subunits of the cytochrome bc1 complex from the bacterium Paracoccus denitrificans were identified by screening a gene library constructed in pBR 322 for expression using a cytochrome c1-specific antibody . These three genes coding for the FeS subunit, cytochrome b, and cytochrome c1 were located on contiguous sites on the genome in a presumed operon arrangement . The DNA-deduced amino acid sequence shows that all three subunits are homologous to corresponding polypeptides of the mitochondrial cytochrome bc1 complex . Cytochrome c1 of Paracoccus is much larger than its mitochondrial counterpart due to an extra 150 amino acids of unique, highly acidic composition; in addition, it is most likely synthesized as a precursor polypeptide. Anal Biochem, 1987 Oct, 166(1), 150 - 7 Use of a chemiluminescence detector for quantitation of nitric oxide produced in assays of denitrifying enzymes; Pai TG et al.; We have developed a closed-flow system that continuously sweeps away gases evolved in enzyme assay mixtures into a commercially available oxides of nitrogen (NOx) analyzer for the quantitation of any nitric oxide present in these gases . The system enabled us to study both the stoichiometry and the kinetics of NO production by a copper-containing nitrite reductase (EC 1.7.99.3) purified from Achromobacter cycloclastes IAM 1013 . In addition to its much greater sensitivity in comparison with standard gas chromatographic (GC) techniques, the method offers the advantage that NO, a very reactive free radical, is immediately swept away and quantitated, obviating the necessity for periodic manipulations and disturbances of the reaction mixture characteristic of other GC quantitations . The characteristics of the system are discussed and its utility in studies of the kinetics and stoichiometry of NO production from nitrite is confirmed by comparison with results obtained using manometric and GC techniques. J Bacteriol, 1987 Oct, 169(10), 4651 - 9 Inhibition of bacteriochlorophyll synthesis in Rhodobacter sphaeroides subsp . denitrificans grown in light under denitrifying conditions; Michalski WP et al.; The inclusion of nitrate or nitrite in cultures of Rhodobacter spaeroides subsp . denitrificans grown heterotrophically in light depressed the formation of bacteriochlorophyll a . The pigment biosynthesis was inhibited at the stage of the reduction of chlorophyllide (chlorin) to bacteriochlorophyllide (tetrahydroporphyrin) since 3-hydroxyethylchlorophyllide a accumulated in the culture medium . The addition of exogenous 5-aminolevulinic acid to these cultures resulted in a complete restoration of bacteriochlorophyll synthesis accompanied by the accumulation of 3-vinylbacteriopheophorbide . This indicates that under these conditions bacteriochlorophyll was formed via an alternative route, in which the reduction of chlorins to tetrahydroporphyrins precedes modifications of the C-3 side chain . The multiple forms of 5-aminolevulinic acid synthase were purified from cells grown with and without nitrate . Antibodies against these proteins were raised in rabbits and used in enzyme-linked immunosorbent assays for various forms of 5-aminolevulinic acid synthase . In denitrifying cells, the amount and activity of fraction I of the enzyme was reduced by approximately 40 and 30%, respectively . Partly active enzymes from both types of cells were activated by cystine trisulfide. J Inorg Biochem, 1987 Oct, 31(2), 143 - 54 EPR of azurins from Pseudomonas aeruginosa and Alcaligenes denitrificans demonstrates pH-dependence of the copper-site geometry in Pseudomonas aeruginosa protein; Groeneveld CM et al.; The X- and Q-band EPR spectra of Pseudomonas aeruginosa (63Cu)azurin and Alcaligenes denitrificans azurin have been measured at pH = 5.2 and 9.2, in the presence and absence of 40% glycerol . The EPR spectra of both proteins could properly be simulated by taking into account a spread in the tetrahedral angle of the copper site . The change in the EPR spectrum of Pseudomonas aeruginosa (63Cu)azurin that is observed upon an increase of the pH from 5.2 to 9.2 is consistent with a small decrease of the average tetrahedral angle from 61 degrees to 60 degrees . This geometrical change is consistent with the interpretation of earlier NMR and EXAFS observations . No pH effect is observed for Alcaligenes denitrificans azurin, in agreement with predictions based on crystallographic evidence . Glycerol has only a marginal effect on the appearance of the EPR spectra, and does not alleviate the "g-strain." Biochem J, 1987 Sep 15, 246(3), 779 - 82 Control of respiration rate in non-growing cells of Paracoccus denitrificans; Kucera I et al.; By means of fluorimetric measurement and by direct determination of intracellular NAD+ and NADH contents, it was proved that the respiration rate of Paracoccus denitrificans cells utilizing glucose is limited by processes preceding NADH oxidation in the respiratory chain, so that the membrane NADH dehydrogenase is not saturated by its substrate . In the separated membrane fraction on saturation with exogenous NADH the main limiting factor is represented by NADH: ubiquinone oxidoreductase. Eur J Biochem, 1987 Sep 15, 167(3), 431 - 9 Subunit II of cytochrome c oxidase from Paracoccus denitrificans . DNA sequence, gene expression and the protein; Steinrucke P et al.; Cytochrome c oxidase from the bacterium Paracoccus denitrificans, while being related to the mitochondrial enzyme in many ways, consists of only two to three different subunits . For the identification of its genes, a Paracoccus DNA library was constructed and screened with specific antibodies for expression of cloned inserts in E . coli . A positive clone expressing immunoreactive products in the molecular mass region of authentic subunit II revealed a high homology of its DNA-deduced amino acid sequence with subunit II sequences of the mitochondrial oxidases; several typical features, such as the transmembrane folding pattern and the presumed copper-binding site, are highly conserved between prokaryotic and mitochondrial polypeptides . A comparison with peptide sequencing data of the purified subunit established the presence of a characteristic N-terminal extension as well as a longer C terminus in the initial translation product of the Paracoccus subunit; by mass spectroscopy, the first N-terminally blocked residue of the mature polypeptide was identified as a pyroglutamate . No code abnormalities, but a highly specific codon usage were observed; no evidence for a localization of the subunit I gene directly adjacent to this gene has been obtained. Arch Microbiol, 1987 Sep, 148(3), 213 - 7 Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads; Tschech A et al.; From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO2 with nitrate as the terminal electron acceptor . All strains grew in defined mineral salts medium; two of them were further characterized . The bacteria were facultatively anaerobic Gram-negative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor . The isolates were tentatively identified as pseudomonads . Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate . While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only . Reduced alicyclic compounds were not degraded . Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate. Appl Environ Microbiol, 1987 Sep, 53(9), 2045 - 9 Loss of N2O reductase activity as an explanation for poor growth of Pseudomonas aeruginosa on N2O; Snyder SW et al.; N2O uptake activity of cells and N2O reductase activity of the soluble fraction from denitrifying bacteria were assayed . Pseudomonas aeruginosa strains PAO1 and P1 lost most of their N2O uptake activity and the ability to grow well on N2O within 2 to 5 h after exposure to N2O . Extensive loss of N2O reductase activity accompanied the nearly complete loss of N2O uptake activity under N2O . Paracoccus denitrificans retained much, but not all, of both activities and the ability to grow vigorously on N2O . The pattern with P . aeruginosa strain P2 resembled that for PAO1 and P1 except that loss of the activities proceeded at a slower rate and growth could continue for up to 12 h after exposure to N2O . The inability of a number of P . aeruginosa strains to grow well on N2O is therefore a direct consequence of the nearly complete loss of N2O reductase activity . Turnover-dependent inactivation of N2O reductase and its reactivation under reducing conditions occurred in vitro for the enzyme from P . aeruginosa and Paracoccus denitrificans . These events may be significant in determining the activity level of N2O reductase in denitrifying bacteria during N2O respiration. J Bacteriol, 1987 Sep, 169(9), 3969 - 75 Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans; Harms N et al.; A genomic clone bank of Paracoccus denitrificans DNA has been constructed in the expression vector set pEX1, pEX2, and pEX3 . Screening of this clone bank with antibodies raised against P . denitrificans methanol dehydrogenase resulted in the isolation of a clone, pNH3, that synthesized methanol dehydrogenase cross-reactive proteins . The nucleotide sequence of the P . denitrificans DNA fragment inserted in this clone has been determined and shown to contain the full methanol dehydrogenase structural gene . DNA cross-hybridization was found with DNA fragments which have been reported to contain the methanol dehydrogenase structural genes from Methylobacterium sp . strain AM1 and Methylobacterium organophilum. Proc R Soc Lond B Biol Sci, 1987 Aug 21, 231(1264), 349 - 58 Growth of Paracoccus denitrificans on {2,3-13C}succinate and {1,4-13C}succinate . II . Isoleucine biosynthesis; Dunstan RH et al.; Paracoccus denitrificans was grown on either {2,3-13C}succinate or {1,4-13C}succinate, and extracts were analysed by using gas chromatography-mass spectrometry . The distribution of label in isoleucine indicated that the 2-ketobutyrate required for isoleucine biosynthesis was mainly produced from pyruvate by 2-keto-acid chain elongation (i.e . the 'pyruvate elongation pathway') . Approximately 10% of isoleucine was produced by a second pathway involving propionyl CoA . Threonine and glutamate were not utilized by P . denitrificans as a source of 2-ketobutyrate production for isoleucine biosynthesis under the growth conditions used. Proc R Soc Lond B Biol Sci, 1987 Aug 21, 231(1264), 339 - 47 Growth of Paracoccus denitrificans on {2,3-13C}succinate and {1,4-13C}succinate . I . The flux of carbon in energy metabolism and the operation of the TCA cycle; Dunstan RH et al.; The metabolism of Paracoccus denitrificans, grown on either {2,3-13C}succinate or {1,4-13C}succinate, was investigated by using gas chromatography-mass spectrometry . The distribution of label in a group of metabolites closely related to the TCA-cycle intermediates showed that the flux of carbon from succinate in energy metabolism in vivo was via pyruvate (malic enzyme) and acetyl CoA . The labelling pattern of the carboxyl groups showed that one fifth of the succinate pool was formed by the regeneration of succinate via the TCA cycle, and four fifths was supplied externally as substrate from the medium. J Mol Biol, 1987 Jul 20, 196(2), 413 - 9 The tertiary structure of azurin from Pseudomonas denitrificans as determined by Cu resonant diffraction using synchrotron radiation; Korszun ZR; The structure of Pseudomonas denitrificans azurin has been solved at 3.0 A resolution by film data collection methods using synchrotron radiation . Bijvoet pairs of reflections were collected using 8.98 keV radiation where both delta f' and delta f", the real and imaginary corrections to azurin's Cu prosthetic group scattering, respectively, are large, and using 8.00 keV radiation where these corrections are smaller . The Cu atom was located by difference Patterson syntheses and used to phase the observed protein structure-factor moduli of selected reflections . An atomic model of the protein was built using a restricted data set and phases for all observed reflections to 3.0 A resolution were subsequently calculated from the initial model . The atomic model was subsequently rebuilt using all observed data . The results of this work are presented here and illustrate the utility of synchrotron radiation phasing techniques in solving the structures of metalloproteins without recourse to multiple isomorphous replacement. J Biol Chem, 1987 Jul 5, 262(19), 9147 - 53 EPR characterization of the iron-sulfur clusters in the NADH: ubiquinone oxidoreductase segment of the respiratory chain in Paracoccus denitrificans; Meinhardt SW et al.; The physicochemical properties of the iron-sulfur clusters present in the NADH:ubiquinone oxidoreductase of Paracoccus denitrificans have been examined in the cytoplasmic membrane particles by redox potentiometry and EPR spectroscopy . Analogous to the iron-sulfur clusters present in the mitochondrial NADH: ubiquinone oxidoreductase, we have found two binuclear and three tetranuclear EPR detectable iron-sulfur clusters, namely, N-1a, N-1b, N-2, N-3, and N-4 . In the bacterial system, the two binuclear clusters differ in line shape and in Em values; the cluster with more rhombic symmetry (gx,y,z = 1.918, 1.937, 2.029) has the Em7.0 value of -150 while the almost axial one (gx,y,z = 1.929, 1.941, 2.019) has Em7.0 of -270 mV . The Em of the former cluster is pH dependent (-60 mV/pH) as in the case of mammalian N-1a while the latter is pH independent as is the mammalian cluster N-1b . The pH-dependent P . denitrificans {2Fe-2S} cluster, which we have labeled N-1a, has an Em7.0 as high as that of N-2, in contrast to the mammalian N-1a . Thus N-1a is reducible with a physiological reductant, NADH in this bacterial system . The Em of the cluster N-2 is also pH dependent (Em7.0 = -130 mV) with a pK value near 7.7 . The Em values of all other clusters exhibit no pH dependence as in the case of their mammalian counterparts . We have found that the cluster N-1a is the most labile component among the five iron-sulfur clusters and may give rise to variable relative spin concentrations and extremely low Em values due to the facile modifications of the microenvironment of the cluster . The P . denitrificans NADH:ubiquinone oxidoreductase provides a unique and useful site I model system where redox composition is similar to the mitochondrial enzyme but with fewer numbers of polypeptides (Yagi, T . (1986) Arch . Biochem . Biophys . 250, 302-311). Ann Inst Pasteur Microbiol, 1987 Jul-Aug, 138(4), 449 - 55 {Denitrifying activity and generation time in 4 species of Rhizobium}; Bourguignon C; Denitrifying capacity and doubling time were measured among 44 strains of Rhizobium belonging to 4 species: B . japonicum, R . lupini, R . meliloti et R . leguminosarum . There was no correlation between doubling time and the rate of production of N2O in the presence of C2H2 in B . japonicum and R . lupini . In 38 other strains, only denitrifying capacity was measured . The percentage of strains showing denitrification capacity varied according to the species: 70% in B . japonicum, 90% in R . lupini, 37% in R . meliloti and 14% in R . leguminosarum . The strains of the latter species showed a very low level of denitrifying activity in comparison with the strains of the 3 other species. Rev Infect Dis, 1987 Jul-Aug, 9(4), 787 - 9 Endocarditis caused by Kingella indologenes; Jenny DB et al.; The first known case of endocarditis caused by Kingella indologenes is reported . A review of the literature reveals only seven cases of endocarditis caused by the other two species of the genus Kingella (Kingella kingae, six cases; Kingella denitrificens, one case) . Kingella organisms appear to be sensitive to a wide variety of antimicrobial agents . The available data suggest that endocarditis caused by Kingella species occurs rarely and is associated with a benign clinical course. J Biochem (Tokyo), 1987 Jul, 102(1), 191 - 7 Purification and properties of dimethylsulfoxide reductase containing a molybdenum cofactor from a photodenitrifier, Rhodopseudomonas sphaeroides f.s . denitrificans; Satoh T et al.; Dimethylsulfoxide (DMSO) reductase was purified to electrophoretic homogeneity from the periplasmic fraction of a photodenitrifier, Rhodopseudomonas sphaeroides f.s . denitrificans . The enzyme had a molecular weight of 82,000 and had no subunit . It contained 1 mol of molybdenum per mol of enzyme, but iron and acid-labile sulfur were not present . The UV-visible spectrum showed only one absorption maximum at 280 nm . Denaturation of the enzyme released a molybdopterin cofactor, the fluorescence spectra of which were almost the same as those of a form B derivative of molybdopterin found in formate dehydrogenase . The Km value for DMSO was 15 microM, which was much lower than that for trimethylamin-N-oxide (TMAO), whereas Vmax with TMAO was larger than that with DMSO. Biochemistry, 1987 Jun 30, 26(13), 4139 - 43 Redox properties of the quinoprotein methylamine dehydrogenase from paracoccus denitrificans; Husain M et al.; Paracoccus denitrificans synthesizes a methylamine dehydrogenase that contains a covalently bound form of pyrroloquinoline quinone as a prosthetic group {Husain, M., & Davison, V.L . (1987) J . Bacteriol . 169, 1712-1717} . Anaerobic reductive titration of this enzyme with dithionite proceeded through a semiquinone intermediate with spectral properties quite distinct from those of the oxidized and reduced species . From these data the molar extinction coefficients were calculated at various wavelengths for the three redox states of this enzyme . The semiquinone was slowly reoxidized under aerobic conditions . The fully reduced enzyme was stable in the presence of oxygen and slowly reoxidized by ferricyanide . Reductive titration of methylamine dehydrogenase with methylamine proceeded directly to the fully reduced form of the enzyme without detectable formation of the semiquinone . Electrochemical titrations of the enzyme yielded an overall midpoint potential value for the two-electron couple (fully oxidized/fully reduced) of 100 +/- 4 mV and an n value of 2.15 +/- 0.15. J Biol Chem, 1987 Jun 25, 262(18), 8702 - 6 Identification of a stable ubisemiquinone and characterization of the effects of ubiquinone oxidation-reduction status on the Rieske iron-sulfur protein in the three-subunit ubiquinol-cytochrome c oxidoreductase complex of Paracoccus denitrificans; Meinhardt SW et al.; The ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans exhibits a thermodynamically stable ubisemiquinone radical detectable by EPR spectroscopy . The radical is centered at g = 2.004, is sensitive to antimycin, and has a midpoint potential at pH 8.5 of +42 mV . These properties are very similar to those of the stable ubisemiquinone (Qi) previously characterized in the cytochrome bc1 complexes of mitochondria . The micro-environment of the Rieske iron-sulfur cluster in the Paracoccus cytochrome bc1 complex changes in parallel with the redox state of the ubiquinone pool . This change is manifested as shifts in the gx, gy, and gz values of the iron-sulfur cluster EPR signal from 1.80, 1.89, and 2.02 to 1.76, 1.90, and 2.03, respectively, as ubiquinone is reduced to ubiquinol . The spectral shift is accompanied by a broadening of the signal and follows a two electron reduction curve, with a midpoint potential at pH 8.5 of +30 mV . A hydroxy analogue of ubiquinone, UHDBT, which inhibits respiration in the cytochrome bc1 complex, shifts the gx, gy, and gz values of the iron-sulfur cluster EPR signal to 1.78, 1.89, and 2.03, respectively, and raises the midpoint potential of the iron-sulfur cluster at pH 7.5 from +265 to +320 mV . These changes in the micro-environment of the Paracoccus Rieske iron-sulfur cluster are like those elicited in mitochondria . These results indicate that the cytochrome bc1 complex of P . denitrificans has a binding site for ubisemiquinone and that this site confers properties on the bound ubisemiquinone similar to those in mitochondria . In addition, the line shape of the Rieske iron-sulfur cluster changes in response to the oxidation-reduction status of ubiquinone, and the midpoint of the iron-sulfur cluster increases in the presence of a hydroxyquinone analogue of ubiquinone . The latter results are also similar to those observed in the mitochondrial cytochrome bc1 complex . However, unlike the mitochondrial complexes, which contain eight to 11 polypeptides and are thought to contain distinct quinone binding proteins, the Paracoccus cytochrome bc1 complex contains only three polypeptide subunits, cytochromes b, c1, and iron-sulfur protein . The ubisemiquinone binding site and the site at which ubiquinone and/or ubiquinol bind to affect the Rieske iron-sulfur cluster in Paracoccus thus exist in the absence of any distinct quinone binding proteins and must be composed of domains contributed by the cytochromes and/or iron-sulfur protein. Biochem J, 1987 Jun 15, 244(3), 661 - 8 Immunochemical probing of the structure and cofactor of NADH dehydrogenase from Paracoccus denitrificans; George CL et al.; Monospecific antibody to the respiratory NADH dehydrogenase from Paracoccus denitrificans was prepared by using as antigen specific immunoprecipitates containing NADH dehydrogenase which were excised from crossed-immunoelectrophoresis plates . The latter were run with selectively solubilized plasma membranes and antibodies against plasma membranes . The antibody immunoprecipitated NADH dehydrogenase from P . denitrificans membranes biosynthetically labelled with 14C and solubilized with a wide range of detergents . All immunoprecipitates contained the two subunits of Mr 48,000 and 25,000, in an approximate 1:1 stoichiometry, that had previously been assigned to NADH dehydrogenase . A polypeptide of Mr 46,000 in P . denitrificans membranes, previously shown to cross-react with a subunit-specific antibody to mitochondrial NADH dehydrogenase (complex I), was not detected in any immunoprecipitate . Under some conditions a third polypeptide, of Mr 31,000, was also detected, but in variable and non-stoichiometric amounts relative to the two other subunits . It was concluded that this polypeptide was incorporated into the immunoprecipitates as an artefact and that the polypeptides of Mr 48,000 and 25,000 are the sole polypeptides firmly identified in the NADH dehydrogenase . Flavoproteins were specifically radiolabelled by growth of P . denitrificans in the presence of {14C}riboflavin . Crossed immunoelectrophoresis of membranes from such cells showed that succinate dehydrogenase contained flavin, but that there was no detectable flavin in NADH dehydrogenase under these conditions . Analysis of excised immunoprecipitates of succinate dehydrogenase showed that flavin was covalently bound to a polypeptide of Mr 56,000 . Flavin was retained by NADH dehydrogenase under mild conditions of detergent solubilization . Subsequent immunoprecipitation, followed by analysis of the acid-extracted flavin, established that FMN is a cofactor, in common with mitochondrial NADH-ubiquinone oxidoreductase (complex I). Eur J Biochem, 1987 Jun 15, 165(3), 665 - 70 Subfractionation and characterization of soluble c-type cytochromes from Paracoccus denitrificans cultured under various limiting conditions in the chemostat; Bosma G et al.; Soluble c-type cytochromes were partially purified from Paracoccus denitrificans cells grown in succinate- and methanol-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures . Five c types could be distinguished with the following apparent molecular masses, absorption maxima and midpoint potentials . (a) 9.2 kDa, 549 nm and +190 mV; (b) 14 kDa, 549 nm and +227 mV; (c) 22 kDa, 552 nm and +190 mV; (d) 30 kDa, 552.7 nm and +160 mV; (e) 45 kDa, a dihaem: 555 nm, +128 mV and 551 nm, -163 mV . The 14-kDa polypeptide was present under all growth conditions examined and most probably is the already well characterized cytochrome c550 . In methanol-limited grown cells three additional cytochromes were found, the 9.2-kDa, 22-kDa and 30-kDa ones . Under oxygen-limited conditions the 45-kDa and under anaerobic growth conditions small quantities of the 30-kDa and 45-kDa cytochromes c were present . Based on the apparent molecular masses the 14-kDa, 22-kDa, 30-kDa and 45-kDa cytochromes may also be present in membrane-fractions. Eur J Biochem, 1987 Jun 15, 165(3), 657 - 63 Isolation and characterization of ubiquinol oxidase complexes from Paracoccus denitrificans cells cultured under various limiting growth conditions in the chemostat; Bosma G et al.; To obtain more information about the composition of the respiratory chain under different growth conditions and about the regulation of electron-transfer to several oxidases and reductases, ubiquinol oxidase complexes were partially purified from membranes of Paracoccus denitrificans cells grown in carbon-source-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures . The isolated enzymes consisted of cytochromes bc1, c552 and aa3 . In comparison with the aerobic ubiquinol oxidase complex, the oxygen- and nitrate-limited ones contained, respectively, less and far less of the cytochrome aa3 subunits and the anaerobic complex also contained lower amounts of cytochrome c552 . In addition, extra haem-containing polypeptides were present with apparent Mr of 14,000, 30,000 and 45,000, the former one only in the anaerobic and the latter two in both the anaerobic and oxygen-limited preparations . This is the first report describing four different membrane-bound c-type cytochromes . The potentiometric and spectral characteristics of the redox components in membrane particles and isolated ubiquinol oxidase fractions were determined by combined potentiometric analysis and spectrum deconvolution . Membranes of nitrate- and oxygen-limited cells contained extra high-potential cytochrome b in comparison with the membranes of aerobically grown cells . No difference was detected between the three isolated ubiquinol oxidase complexes . Aberrances with already published values of redox potentials are discussed. Arch Microbiol, 1987 Jun, 148(1), 20 - 4 Immunochemical patterns of distribution of nitrous oxide reductase and nitrite reductase (cytochrome cd1) among denitrifying pseudomonads; Korner H et al.; The novel multicopper enzyme nitrous oxide reductase from Pseudomonas perfectomarina was purified to homogeneity to study its properties and distribution in various pseudomonads and other selected denitrifying genera by immunochemical techniques . Quantitation of immunochemical crossreactivity by micro-complement fixation within the denitrifying pseudomonads of Palleroni's ribosomal ribonucleic acid group I corresponded to the taxonomic positions established by nucleic acid hybridization . The assignment of P . perfectomarina to the stutzerigroup (as strain ZoBell) was consolidated by immunochemical crossreactivity based on nitrous oxide reductase . Crossreactivity of nitrite reductase (cytochrome cd1) with a respective P . perfectomarina rabbit antiserum was limited to strain DSM 50227 of P . stutzeri; although it could not contribute information towards broader relationships within rRNA group I, it lent further prove to the unity of these two species. Biochemistry, 1987 May 19, 26(10), 2822 - 8 Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms; Yagi T; The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) . This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain . Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH . By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and Bacillus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD . In the bovine and P . denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase . In the bovine NADH-ubiquinone reductase complex (complex I), {14C}DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000 . The time course of labeling of the 29,000 molecular weight subunit with {14C}DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity. Biochemistry, 1987 May 19, 26(10), 2711 - 22 Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins; Petrich JW et al.; We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade) . Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments . From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale . The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale . The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II) . The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor . The involvement of protein matrix and donor mobility for electron transfer is discussed . The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood . The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics. J Biol Chem, 1987 May 15, 262(14), 6515 - 25 Purification and some characteristics of nitrous oxide reductase from Paracoccus denitrificans; Snyder SW et al.; Nitrous oxide reductase from the denitrifying bacterium Paracoccus denitrificans has been purified very nearly to homogeneity by an anaerobic procedure that results in a product with high specific activity . The enzyme is a dimer of about Mr 144,000 composed of two subunits of apparently equal Mr and contains 4 mol of Cu per mol of subunit . The isoelectric point is 4.3; specific activity at 25 degrees C, pH 7.1, is 122 mumol X min-1 X mg of protein-1; and Km is about 7 microM N2O under the same conditions . The N2O- and O2-oxidized forms of the enzyme had principal absorption bands at 550 and 820 nm; the dithionite-reduced form, at 650 nm . The extinction coefficient at 550 nm for the oxidized enzyme is about 5300 (M subunit)-1 X cm-1 . Ferricyanide-oxidized enzyme and enzyme exposed to O2 for a couple of days at 4 degrees C exhibited additional bands at 480, 620, and 780 nm and had very low specific activities . Cu-EPR signals were observed with oxidized and reduced forms of the enzyme with g perpendicular values at 2.042 and 2.055, respectively . The O2-oxidized enzyme had g parallel and A parallel values of about 2.244 and 35 gauss, respectively, based on the observation of four hyperfine lines in the g parallel region . The enzyme may therefore contain at least one Cu atom approximating the "Type 1" class . Spin counts against Cu-EDTA standards suggest that 20-30% of the enzyme-bound Cu is EPR detectable in the O2-oxidized enzyme and 7-15% in the enzyme as prepared and in the reduced enzyme . Much of the Cu thus appears to be EPR silent . Nitrous oxide reductase was observed to undergo turnover-dependent inactivation, and nitrite and fluoride among other anions were found to accelerate this process . In a number of characteristics, the enzyme resembles nitrous oxide reductase recently purified from Pseudomonas perfectomarina and Rhodopseudomonas sphaeroides, particularly the former . Some differences appear related to whether or not purification is carried out entirely under anaerobic conditions. Ann Inst Pasteur Microbiol, 1987 May-Jun, 138(3), 371 - 83 Denitrification by a marine bacterium Pseudomonas nautica strain 617; Bonin P et al.; A bacterial strain was isolated from a marine sediment highly contaminated by hydrocarbons . From taxonomic tests, it was identified as Pseudomonas nautica . This marine strain was able to grow on nitrate, nitrite and nitrous oxide as an electron acceptor . The terminal product from the denitrification was dinitrogen . Thus, P . nautica was a denitrifier . The kinetics of each step of denitrification was examined in resting cell suspensions . The relative rates of nitrate and nitrite reduction and of nitrite reduction and nitrous oxide production explain, respectively, the presence of accumulated nitrite and that of compound intermediate between nitrite and nitrous oxide. J Biochem (Tokyo), 1987 May, 101(5), 1129 - 39 Cytochrome P-450 related to P-4504 from phenobarbital-treated rabbit liver: molecular cloning of cDNA and characterization of cytochrome P-450 obtained by its expression in yeast cells; Imai Y; cDNA complementary to mRNA coding for a minor form of cytochrome P-450 from phenobarbital-treated rabbit liver (pHP3) was isolated using cDNA for the major phenobarbital-inducible cytochrome P-450 of rat liver as a probe in the first screening of a cDNA library . The nucleotide sequence of pHP3 was determined and contained a continuous reading frame encoding 490 amino acids . The deduced amino acid sequence of pHP3 protein exhibited about 50% homology with the major cytochrome P-450 from phenobarbital-treated rabbit liver, while the homology was as high as 80% between two minor cytochrome forms, pHP2 and pHP3 . Two expression plasmids, pAHF3 and pAH delta N3, were constructed by insertion of pHP3 fragment between yeast alcohol dehydrogenase 1 (ADH1) promoter and terminator regions . pAHF3 contained the entire coding sequence of pHP3, but nucleotide sequences for the N-terminal region of pHP3 protein (from the 2nd to the 3rd amino acid) were deleted in pAH delta N3 . On introduction of the constructed plasmids into Saccharomyces cerevisiae AH22 cells, the absorption spectrum of cytochrome P-450 was detected in the microsomal fraction from the transformed cells carrying pAHF3 . On the other hand, cytochrome P-450 could not be detected spectrophotometrically in any subcellular fractions from the yeast cells carrying pAH delta N3, although the transcript of pHP3 insert was detected in RNA blot analysis . These results suggest that the N-terminal region of pHP3 protein plays an important role in accumulation of the newly synthesized pHP3 protein in yeast cells . Cytochrome P-450 (pHP3) was solubilized from microsomal membranes of the transformed yeast cells and purified partially on an aminooctyl Sepharose column (specific content, about 6 nmol per mg of protein) . In the oxidized state the cytochrome preparation exhibited an absorption spectrum characteristic of a low-spin ferric cytochrome P-450 . The reduced CO complex of the cytochrome showed a Soret absorption maximum at 450 nm . The monooxygenase activity of cytochrome P-450 (pHP3) was examined in a reconstituted system containing the cytochrome preparation and NADPH-cytochrome P-450 reductase . Cytochrome P-450 (pHP3) catalyzed N-demethylation of benzphetamine and aminopyrine and denitrification of 1-nitropropane . Addition of cytochrome b5 to the reconstituted system resulted in stimulation of the N-demethylation activities but inhibition of the denitrification activity . Neither 7-ethoxycoumarin O-deethylation activity nor acetanilide p-hydroxylation activity was detected, either in the presence or absence of cytochrome b5.(ABSTRACT TRUNCATED AT 400 WORDS) Eur J Biochem, 1987 Apr 15, 164(2), 295 - 300 Cytochrome c oxidase is a three-copper, two-heme-A protein; Steffens GC et al.; Metal contents of preparations of procaryotic (Paracoccus denitrificans) and eucaryotic (beef heart) cytochrome c oxidases have been determined by inductively coupled plasma atomic emission spectroscopy and shown to be stoichiometrically related to the protein contents . The results show that oxidases which possess subunits I and II have three copper atoms besides hemes a and a3 (Paracoccus denitrificans, Cu: 2.97 +/- 0.08 and Fe: 2.09 +/- 0.10; bovine heart, Cu: 2.83 +/- 0.07 and Fe: 1.94 +/- 0.12) . Together with data reported for the c1 aa3 oxidase from Thermus thermophilus, the following conclusions can be drawn . Subunit I binds two copper atoms and both hemes a and a3 and thus is the universal terminal oxidase of this spectral type . Subunit II binds one copper and functions as an electron conductor . The mitochondrial respiratory complex IV binds, in addition to three copper and two hemes a, stoichiometric amounts of magnesium and zinc (bovine heart Mg: 0.98 +/- 0.05 and Zn: 1.01 +/- 0.04). FEBS Lett, 1987 Apr 6, 214(1), 75 - 80 Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III; Fink PS et al.; A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector . A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases . Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions. Appl Environ Microbiol, 1987 Apr, 53(4), 810 - 5 Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro-, 4-bromo-, and 4-iodobenzoate by Alcaligenes denitrificans NTB-1; van den Tweel WJ et al.; Alcaligenes denitrificans NTB-1, previously isolated on 4-chlorobenzoate, also utilized 4-bromo-, 4-iodo-, and 2,4-dichlorobenzoate but not 4-fluorobenzoate as a sole carbon and energy source . During growth, stoichiometric amounts of halide were released . Experiments with whole cells and cell extracts revealed that 4-bromo- and 4-iodobenzoate were metabolized like 4-chlorobenzoate, involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoate, which in turn was hydroxylated to 3,4-dihydroxybenzoate . The initial step in the metabolism of 2,4-dichlorobenzoate was catalyzed by a novel type of reaction for aerobic organisms, involving inducible reductive dechlorination to 4-chlorobenzoate . Under conditions of low and controlled oxygen concentrations, A . denitrificans NTB-1 converted all 4-halobenzoates and 2,4-dichlorobenzoate almost quantitatively to 4-hydroxybenzoate. J Bacteriol, 1987 Apr, 169(4), 1712 - 7 Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans; Husain M et al.; Methylamine dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methylamine-grown cells . The enzyme exhibited a pI value of 4.3 and was composed of two 46,700-dalton subunits and two 15,500-dalton subunits . Each small subunit possessed a covalently bound pyrrolo-quinoline quinone prosthetic group . The amino acid compositions of the large and small subunits are very similar to those of other methylamine dehydrogenases which have been isolated from taxonomically different sources . The enzyme was able to catalyze the oxidation of a wide variety of primary aliphatic amines and diamines, but it did not react with secondary, tertiary, or aromatic amines . The enzyme exhibited optimal activity at pH 7.5, with Km values of 12.5 microM for methylamine and 156 microM for phenazine ethosulfate and a Vmax of 16.9 mumol/min per mg of protein . No loss of enzyme activity was observed after incubation for 48 h at pH values ranging from 3.0 to 10.5, and the enzyme was very stable to thermal denaturation . Enzyme activity and immunological detection of each subunit were only observed with cells which had been grown on methylamine as a carbon source. Appl Environ Microbiol, 1987 Apr, 53(4), 745 - 50 Oxygen regulation of nitrate uptake in denitrifying Pseudomonas aeruginosa; Hernandez D et al.; Oxygen had an immediate and reversible inhibitory effect on nitrate respiration by denitrifying cultures of Pseudomonas aeruginosa . Inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake, since nitrate reduction to nitrite in cell extracts was not affected by oxygen . The degree of oxygen inhibition was dependent on the concentration of oxygen, and increasing nitrate concentrations could not overcome the inhibition . The inhibitory effect of oxygen was maximal at approximately 0.2% oxygen saturation . The inhibition appeared to be specific for nitrate uptake . Nitrite uptake was not affected by these low levels of aeration, and nitrite reduction was only partially inhibited in the presence of oxygen . The regulation of nitrate respiration at the level of transport by oxygen may represent a major mechanism by which the entire denitrification pathway is regulated in P . aeruginosa. J Biochem (Tokyo), 1987 Apr, 101(4), 957 - 66 Complete amino acid sequence of cytochrome c551 from Erythrobacter species strain OCh 114; Okamura K et al.; The complete amino acid sequence of cytochrome c551 isolated from an aerobic photosynthetic bacterium, Erythrobacter sp . strain OCh 114, was determined . The cytochrome molecule was composed of a total of 119 amino acid residues and its molecular weight including heme was calculated to be 13,235 . The sequence was (Sequence: see text) . Its molecular weight indicates that this cytochrome is of the L-type . Sequence alignment with other bacterial cytochromes c shows that this cytochrome is similar to cytochromes c of Rhodobacter capsulatus, Rhodobacter sphaeroides, and Paracoccus denitrificans, which were grouped into the alpha-3 subcluster from the 16S rRNA sequence analysis. Biochim Biophys Acta, 1987 Feb 25, 911(3), 334 - 40 Detergent inhibition of nitric-oxide reductase activity; Shapleigh JP et al.; Gas chromatography revealed that exposure of extracts of the denitrifiers 'Achromobacter cycloclastes', Paracoccus denitrificans, Pseudomonas aeruginosa and Pseudomonas perfectomarina to Triton X-100 inhibited reduction of NO to N2O, and thus concomitantly inhibited reduction of NO2- to N2O . After exposure of extracts to Triton X-100, the ratio of H+ consumed to NO2- added decreased from approx . 2.0 (for untreated extracts) to approx . 1.5, which indicated that NO2- was reduced to NO by the treated extracts . Addition of a CHAPS-soluble extract (devoid of nitrite reductase activity but rich in nitric-oxide reductase activity) to the Triton X-100-treated extract of P . denitrificans restored capacity for reduction of NO2- on to N2O . Exposure to either the NO that accumulated from reduction of NO2- or to enthetic NO transiently inhibited rates of NO2- reduction in Triton X-100-treated extracts . Use of an Oxides of Nitrogen analyzer indicated that only 5-33% of NO2- reduced by untreated extracts appeared in the stripping gas as NO, whereas 80-95% of NO2- reduced by Triton X-100-treated extracts was recovered as NO. Arch Biochem Biophys, 1987 Feb 15, 253(1), 199 - 204 Is the ubiquinone pool in the respiratory chain of the bacterium Paracoccus denitrificans really unhomogeneous? Kucera I, Kozak L, Dadak V. We have established the participation of a mobile redox pool in the respiratory chain of anaerobically grown bacterium Paracoccus denitrificans . In testing the kinetical homogeneity of the pool it was found that the ratio of fluxes of electron transport toward the terminal acceptors oxygen and nitrate was coincident for the respiratory substrates NADH and succinate; this provides evidence against the preferential link of one dehydrogenase with a distinct terminal enzyme through the separate pool of ubiquinone . The deviation from the expected behavior observed in comparing the titration of NADH oxidase and succinate oxidase with respiratory inhibitors such as mucidin (inhibitor in the bc1 region) or cyanide can be accounted for by the activation of succinate dehydrogenase upon the increase in the reduced state of respiratory components during the titration. Biochemistry, 1987 Feb 10, 26(3), 758 - 64 Laser flash photolysis studies of electron transfer between ferredoxin-NADP+ reductase and several high-potential redox proteins; Bhattacharyya AK et al.; Complex formation and the kinetics of electron transfer between ferredoxin-NADP+ reductase (FNR) and two structurally homologous acidic 4Fe-4S high-potential ferredoxins (HiPIP's) from Ectothiorhodospira halophila (HP1 and HP2) and two structurally homologous cytochromes c2 from Paracoccus denitrificans and Rhodospirillum rubrum (PC2, and RC2, respectively) have been investigated by gel filtration and laser flash photolysis techniques . Gel filtration studies indicated that complex formation occurred between FNRox and HP1ox or HP2ox at low ionic strength (10 mM) and that the complexes were completely dissociated at high ionic strength (310 mM) . Laser flash photolysis using lumiflavin as the reductant demonstrated that both free HP1ox and HP2ox reacted primarily with the anionic form of fully reduced lumiflavin (LFH-), whereas FNR was unreactive . Second-order rate constants of 1 X 10(6) and 0.8 X 10(6) M-1 s-1 were obtained for these reactions at 10 mM ionic strength . Increasing the ionic strength to 310 mM resulted in an approximately 1.5-fold increase in the rate constant . Inclusion of stoichiometric amounts of FNRox into the reaction mixture at low ionic strength led to a 2.5-fold increase in the rate constants . The reaction of 5-deazariboflavin semiquinone (5-dRf.) with the oxidized HiPIP's was also investigated by laser flash photolysis . Second-order rate constants of 3.0 X 10(8) M-1 s-1 (HP1) and 2.5 X 10(8) M-1 s-1 (HP2) were obtained for the free proteins at 10 mM ionic strength . Under the same conditions, 5-dRf . reacted with free FNRox, resulting in the formation of the neutral protein-bound semiquinone (FNR.), with a second-order rate constant of 6 X 10(8) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1987 Feb, 169(2), 874 - 9 Mechanism of nitrogenase switch-off by oxygen; Goldberg I et al.; Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f . sp . denitrificans and Rhodopseudomonas capsulata strains AD2 and BK5) . In K . pneumoniae 50% inhibition of acetylene reduction was attained at an O2 concentration of 0.37 microM . Cyanide (90 microM), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O2 concentration that caused 50% inhibition of nitrogenase activity to 2.9 microM . A mutant strain of K . pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but its nitrogenase activity is similar to wild-type activity . Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 microM O2 . Inhibition by CN- of 40 to 50% of the O2 uptake in the mutant shifted the O2 concentration that caused 50% inhibition of nitrogenase to 1.58 microM . Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase . Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors . Addition of 2 mM sulfite to cell suspensions of R . capsulata B10 and R . sphaeroides inhibited nitrogenase activity . Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R . sphaeroides but not in R . capsulata B10, which is not capable of enzymatic reduction of NO2- . Lower concentrations of NO2- were required to inhibit the activity in NO3- -grown cells, which have higher activities of nitrite reductase.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1987 Jan 13, 26(1), 71 - 82 Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans; Ainscough EW et al.; Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans . These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins . The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins . Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5 . A small reversible change occurs at pH approximately 11.4 . In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks . Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112 . Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively . The redox potential of A . denitrificans azurin at pH 7.0, Eo', is 276 mV . These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45 . Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions . Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin {with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin} . On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups. J Clin Microbiol, 1987 Jan, 25(1), 37 - 41 Clinical evaluation of the Vitek Neisseria-Haemophilus Identification card; Janda WM et al.; A clinical evaluation of the Vitek Neisseria-Haemophilus Identification (NHI) card (Vitek Systems, Inc., Hazelwood, Mo.) was performed with 480 clinical isolates and stock strains of Neisseria spp., Haemophilus spp., and other fastidious microorganisms included in the data base of the system . Identifications obtained with the NHI card were compared with those determined by conventional methods . The card identified 83.2% of 244 Neisseria spp . and Branhamella catarrhalis, 54.9% of 164 Haemophilus spp., and 84.7% of 72 fastidious gram-negative species with no further testing required . Some isolates produced good confidence-marginal separation identifications, in which the correct identification was listed with one or two other possible identifications and extra tests were required and suggested . When isolates producing good confidence-marginal separation identifications were included, correct identifications of these organism groups increased to 97.1, 92.7, and 94.4%, respectively . Among the commonly isolated microorganisms, the NHI card identified 99.1% of 110 N . gonorrhoeae, 98.5% of 68 N . meningitidis, 93.9% of 98 H . influenzae, and 95.6% of 46 H . parainfluenzae strains . All of these organisms produced excellent to very good confidence level identifications except for H . influenzae biotypes II, III, and VII, for which hemolytic reactions were required for differentiation from H . haemolyticus . The NHI card reliably identified other fastidious gram-negative species, including H . aphrophilus, Eikenella corrodens, Gardnerella vaginalis, and Kingella denitrificans. Gene, 1987, 52(2-3), 147 - 54 Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria; Fellay R et al.; We have constructed a series of derivatives of the omega interposon {Prentki and Krisch, Gene 29 (1984) 303-313} that can be used for in vitro insertional mutagenesis . Each of these DNA fragments carries a different antibiotic or Hg2+ resistance gene (ApR, CmR, TcR, KmR or HgR) which is flanked, in inverted orientation, by transcription and translation termination signals and by synthetic polylinkers . The DNA of these interposons can be easily purified and then inserted, by in vitro ligation, into a plasmid linearized either at random by DNase I or at specific sites by restriction enzymes . Plasmid molecules which contain an interposon insertion can be identified by expression of its drug resistance . The position of the interposon can be precisely mapped by the restriction sites in the flanking polylinker . To verify their properties we have used these omega derivatives to mutagenize a broad host range plasmid which contains the entire meta-cleavage pathway of the toluene degradation plasmid pWW0 of Pseudomonas putida . Insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli . We also show that when a plasmid containing an omega interposon is transferred by conjugal mobilization from E . coli to P . putida, Agrobacterium tumefaciens, Erwinia chrysanthemi, Paracoccus denitrificans or Rhizobium leguminosarum, the appropriate interposon drug resistance is usually expressed and, compared to the non-mutated plasmid, much reduced levels of C23O activity are detected . Thus, the selection and/or characterization of omega insertional mutations can be carried out in these bacterial species. J Appl Bacteriol, 1986 Dec, 61(6), 491 - 7 O-demethylation, dehydroxylation, ring-reduction and cleavage of aromatic substrates by Enterobacteriaceae under anaerobic conditions; Grbic-Galic D; Four fermentative facultative anaerobes, members of the genera Enterobacter and Escherichia, were tested for their ability to transform an aromatic lignin derivative, 3-methoxy-4-hydroxy-cinnamic acid (ferulic acid), under anaerobic (fermentative) conditions . The pure cultures studied were shown to O-demethylate, dehydroxylate, reduce the double bond in the side-chain, decarboxylate the aromatic ring to the stage of benzoate and to reduce the ring to an alicyclic acid . Aromatic hydrocarbons (toluene, ethylbenzene and propylbenzene), as well as phenols (phenol, o-cresol, p-cresol, 2-ethylphenol and 3-hydroxy-4-ethylphenol) were also produced . In addition, during 3 months incubation, the cleavage of the aromatic ring occurred, whereby a small fraction of the substrate was converted to straight-chain and branched (methylated, ethylated) five- to eight-carbon aliphatic acids . The results indicate that pure cultures of fermentative facultative anaerobes might be capable of degrading substituted aromatic acids to aliphatic products under strictly anaerobic (fermentative) conditions . These abilities, which have so far been found only in denitrifying pseudomonads among facultative anaerobes, might be common in Enterobacteriaceae . It is conceivable that these bacteria are important as degraders of aromatic compounds in anaerobic ecosystems. Appl Environ Microbiol, 1986 Nov, 52(5), 1117 - 22 Anaerobic oxidation of p-cresol by a denitrifying bacterium; Bossert ID et al.; Metabolism of p-cresol (pCr) under nitrate-reducing conditions is mediated by the denitrifying bacterial isolate PC-07 . The methyl substituent of the substrate is oxidized anaerobically by whole-cell suspensions of PC-07 through a series of dehydrogenation and hydration reactions to yield p-hydroxybenzoate (pOHB) in stoichiometric proportions . The partially oxidized intermediates in the pathway p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde can also serve as substrates for pOHB formation . Nitrate is required as the external electron acceptor and is reduced to molecular N2 . Reduction of the nitrate is stoichiometric, with pCr serving as the electron donor . In addition, the molar relationship between the electron acceptor (NO3-) reduced to the electron donor oxidized decreased to approximately 2:3 and then to 1:3 when p-hydroxybenzyl alcohol or p-hydroxybenzaldehyde, respectively, served as substrates . The decreased ratios were to be expected when the partially oxidized intermediates served as substrates, because they provided correspondingly less reducing power for pOHB formation . The anaerobic oxidation of pCr by PC-07 demonstrates a mechanism whereby aromatic compounds can be transformed in anoxic environments. J Clin Microbiol, 1986 Nov, 24(5), 879 - 81 Peritonitis caused by Alcaligenes denitrificans subsp . xylosoxydans: case report and review of the literature; Morrison AJ Jr et al.; We report the third human case of peritonitis caused by Alcaligenes denitrificans subsp . xylosoxydans and review the English literature regarding community-acquired and nonsocomial infection and colonization that results from this bacterium . The biochemical and genetic characteristics supporting the pathogenic potential of A . denitrificans subsp . xylosoxydans are reviewed, and the antimicrobial susceptibility profile of the organism is summarized. Arch Biochem Biophys, 1986 Nov 1, 250(2), 302 - 11 Purification and characterization of NADH dehydrogenase complex from Paracoccus denitrificans; Yagi T; An NADH dehydrogenase complex was isolated from the plasma membranes of aerobically grown Paracoccus denitrificans cells by extraction with NaBr and purification on an NAD-agarose column . The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase complex was about 10 times higher than that of the NaBr extract . The preparation was composed of 10 (6 major and 4 minor) unlike polypeptides, and lacked identifiable components and activities characteristic of other enzyme complexes of the oxidative phosphorylation system . The purified enzyme contained noncovalently bound FMN, nonheme iron, and acid-labile sulfide . The ratio of FMN to nonheme iron to acid-labile sulfide was 1:13 approximately 14:11 approximately 12, suggestive of the presence of multiple iron-sulfur clusters . The isolated NADH dehydrogenase complex cross-reacted with antisera to beef heart mitochondrial complex I and protein fraction derived therefrom, indicating the presence in the Paracoccus enzyme of antigenic sites similar to those in the intact complex I and its iron-sulfur protein and possibly hydrophobic protein fractions. J Biochem (Tokyo), 1986 Nov, 100(5), 1261 - 7 Evolutionary relationship of denitrifying bacteria as deduced from 5S rRNA sequences; Ohkubo S et al.; The nucleotide sequences of 5S rRNA from seven denitrifying bacteria have been determined . Based on these sequences and those reported in the literature (including two denitrifiers), a phylogenic tree of 104 eubacterial 5S rRNA sequences has been constructed to establish the position of the denitrifying bacteria . These bacteria belong to either one of the three major subgroups of gram-negative bacteria . The grouping based on 5S rRNA sequences is almost compatible with the type of the nitrite reductases, with the one apparent exception of Paracoccus denitrificans ATCC 13543 . Moreover, the separation time of most of the denitrifying bacteria from other non-denitrifying bacteria belonging to the same subgroup is recent . These results suggest that the denitrifying systems in these bacteria would have developed polyphyletically, and not so anciently, during eubacterial evolution. FEBS Lett, 1986 Oct 27, 207(2), 239 - 42 Measurement of the oxidation-reduction potentials of amicyanin and c-type cytochromes from Paracoccus denitrificans; Gray KA et al.; The oxidation-reduction potentials of four periplasmic electron carrier proteins from Paracoccus denitrificans have been determined . Their midpoint potentials are: amicyanin, 294 +/- 6 mV; cytochrome c-550, 253 +/- 5 mV; cytochrome c-551i, 190 +/- 4 mV; and cytochrome c-553i, 148 +/- 5 mV . Although rapid amicyanin-mediated transfer of electrons from methylamine dehydrogenase to cytochrome c-551i was observed, reduced amicyanin did not reduce oxidized cytochrome c-551i in the absence of methylamine dehydrogenase. Biochemistry, 1986 Oct 21, 25(21), 6502 - 7 Electron-transfer reactions between flavodoxin semiquinone and c-type cytochromes: comparisons between various flavodoxins; Cheddar G et al.; As an extension of previous work from this laboratory using Clostridium pasteurianum flavodoxin {Tollin, G., Cheddar, G., Watkins, J . A., Meyer, T . E., & Cusanovich, M . A . (1984) Biochemistry 23, 6345-6349}, we have measured the rate constants as a function of ionic strength for electron transfer from the semiquinones of Clostridium MP, Anacystis nidulans, and Azotobacter vinelandii flavodoxins to the following oxidants: cytochrome c from tuna and horse, Paracoccus denitrificans cytochrome c2, Pseudomonas aeruginosa cytochrome c-551, and ferricyanide . The rate constants extrapolated to infinite ionic strength (k infinity) for the C . MP flavodoxin are all slightly smaller than for the C . pasteurianum flavodoxin, as would be predicted on the basis of the higher redox potential of the C . MP protein . This indicates that there is a close similarity between the surface topographies of the two proteins in the vicinity of the coenzyme binding site . Moreover, the electrostatic interactions between the two flavodoxins and the various oxidants are also approximately the same . These studies justify our previous use of the crystallographic structure of the C . MP flavodoxin to interpret kinetic results obtained with the structurally uncharacterized C . pasteurianum flavodoxin . Despite their lower redox potentials, both Anacystis and Azotobacter flavodoxins are appreciably less reactive toward all of these oxidants (as much as 2 orders of magnitude in some cases) than are the Clostridium flavodoxins.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1986 Oct, 168(1), 455 - 7 Iron-dependent production of a heat-modifiable, 23,000-Mr outer membrane protein in Paracoccus denitrificans; Wee S et al.; Production of a 23,000-Mr major outer membrane protein of Paracoccus denitrificans ATCC 13543 was dependent upon the addition of iron to a succinate-salts medium . The 23,000-Mr protein was not produced in an iron-deficient medium, but production of five outer membrane proteins in the 85,000- to 72,000-Mr range and of catechol were induced . The 23,000-Mr protein was not produced in a complex medium even when ferric citrate was added to the medium . Production of the protein was influenced by the carbon source and was decreased by peptone. J Gen Microbiol, 1986 Oct, 132 ( Pt 10), 2709 - 21 Pseudomonas fluorescens biovar V: its resolution into distinct component groups and the relationship of these groups to other P . fluorescens biovars, to P . putida, and to psychrotrophic pseudomonads associated with food spoilage; Barrett EL et al.; A numerical taxonomic analysis was performed to evaluate the appropriateness of a single biovar designation (biovar V) for all Pseudomonas fluorescens isolates negative for denitrification, levan production and phenazine pigmentation and to determine the relationship of biovar V strains to other taxa within the same Pseudomonas RNA homology group . Seventy-two strains assigned to P . fluorescens biovar V and four strains of P . fragi were characterized and the data subjected to a numerical taxonomic analysis along with comparable data for 17 previously characterized strains of this biovar and 89 P . putida strains . Seven distinct biovar V clusters containing three or more strains were revealed, and the carbon sources useful for their differentiation were identified . Cluster 1 (38 strains) closely resembled two atypical P . fluorescens I strains . It was also related to P . fluorescens biovar IV and to P . fragi . Cluster 2 (5 strains) was related to cluster 1 . Cluster 3 (7 strains) was identical to a major group of meat spoilage psychrotrophic pseudomonads (P . lundensis) . Cluster 4 (3 strains) was not related to any other group examined . Cluster 5 consisted of six isolates initially designated P . putida A along with four P . fluorescens biovar V strains all of which resembled P . putida more than they resembled the other P . fluorescens groups . Cluster 6 (16 strains) was distinct from the other biovar V clusters, but was closely related to P . fluorescens biovars I and II . Cluster 7 (3 strains) shared many characteristics with cluster 5 . Separate P . fluorescens biovar designations are proposed for cluster 6 and for the combined clusters 1 and 2 . A new P . putida biovar is proposed for the combined clusters 5 and 7. FEBS Lett, 1986 Sep 29, 206(1), 154 - 6 A gene in Paracoccus for subunit III of cytochrome oxidase; Saraste M et al.; The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa3) subunits are located has been cloned . DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme . This subunit is absent from the isolated Paracoccus oxidase . It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane . This may explain the observed discrepancies in the function of the isolated enzyme. J Biol Chem, 1986 Sep 15, 261(26), 12282 - 9 Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans; Yang XH et al.; A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract . The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein . Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b . The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin . The Paracoccus bc1 complex consists of only three polypeptide subunits . On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa . The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively . The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex . The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1 . This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain . The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria . The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date . These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle. J Biochem Toxicol, 1986 Sep, 1(3), 71 - 83 Cytochrome P-450-mediated denitrification of 2-nitropropane in mouse liver microsomes; Marker EK et al.; Enzymatic denitrification of 2-nitropropane (2NP) was investigated in an NADPH-dependent hepatic microsomal system from male CD1 mice . The involvement of cytochrome P-450 (P-450) as the catalyst in 2NP denitrification was revealed by the induction of nitrite-releasing activity following phenobarbital (PB) pretreatment, by a decrease in activity with carbon tetrachloride pretreatment, by the inhibition of the reaction with classical P-450 inhibitors, and by the observation of a type I binding spectrum . Under optimal conditions, two pH-dependent peaks of activity were observed at pH 7.6 and pH 8.8, each with its own optimal substrate concentration . Inhibition of the reaction by metyrapone and carbon monoxide (CO) (among others) produced differential responses dependent on pH . These results, along with two pH optima and two substrate optima, suggested the involvement of multiple P-450 isozymes . Average specific activities were 8.05 nmoles of nitrite released per minute per milligram microsomal protein at pH 7.6 and 6.44 nmoles of nitrite released per minute per milligram microsomal protein at pH 8.8 . Acetone was identified as the second product of the reaction by gas chromatography/mass spectrometry (GC/MS) . Stoichiometry studies indicated that the acetone production was slightly less than expected (about 70%) from nitrite release . Up to 25% residual activity was observed under anaerobic conditions . These results suggested that though the predominant reaction mechanism was oxidative, oxygen-independent metabolism of 2NP also occurred to some extent . In contrast to the reported lack of activity in untreated rat, the observed denitrification in uninduced mouse liver microsomes was significant and suggested that major species-specific differences exist in the in vitro metabolism of 2NP. Appl Environ Microbiol, 1986 Aug, 52(2), 262 - 8 Microbial release of 226Ra2+ from (Ba,Ra)SO4 sludges from uranium mine wastes; Fedorak PM et al.; 226Ra2+ is removed from uranium mine effluents by coprecipitation with BaSO4 . (Ba,Ra)SO4 sludge samples from two Canadian mine sites were found to contain active heterotrophic populations of aerobic, anaerobic, denitrifying, and sulfate-reducing bacteria . Under laboratory conditions, sulfate reduction occurred in batch cultures when carbon sources such as acetate, glucose, glycollate, lactate, or pyruvate were added to samples of (Ba,Ra)SO4 sludge . No external sources of nitrogen or phosphate were required for this activity . Further studies with lactate supplementation showed that once the soluble SO4(2-) in the overlying water was depleted, Ba2+ and 226Ra2+ were dissolved from the (Ba,Ra)SO4 sludge, with the concurrent production of S2- . Levels of dissolved 226Ra2+ reached approximately 400 Bq/liter after 10 weeks of incubation . Results suggest that the ultimate disposal of these sludges must maintain conditions to minimize the activity of the indigenous sulfate-reducing bacteria to ensure that unacceptably high levels of 226Ra2+ are not released to the environment. J Bioenerg Biomembr, 1986 Aug, 18(4), 277 - 84 Cytochrome c oxidase from Paracoccus denitrificans in Triton X-100: aggregation state and kinetics; Bolli R et al.; Cytochrome c oxidase from Paracoccus denitrificans was homogeneously dispersed in Triton X-100 . Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined . After subtraction of the bound detergent (111 mol/mol heme aa3) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits . This monomer showed high cytochrome c oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (Vmax = 85 s-1) . The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochrome c concentration . A Km of 3.6 X 10(-6) M was evaluated, similar to the Km observed in the presence of dodecyl maltoside {Nalecz et al . (1985). J Biol Chem, 1986 Jul 25, 261(21), 9652 - 6 Isotope labeling studies on the mechanism of N-N bond formation in denitrification; Aerssens E et al.; The mechanism of the denitrification and nitrosation reactions catalyzed by the heme cd-containing nitrite reductase from Pseudomonas stutzeri JM 300 has been studied with whole cell suspensions using H2(18)O, 15NO, and 15NO-2 . The extent of H2(18)O exchange with the enzyme-bound nitrosyl intermediate, as determined by the 18O content of product N2O, decreased with increasing nitrite concentration, which is consistent with production of N2O by sequential reaction of two nitrite ions with the enzyme . Reaction of NO with whole cells in H2(18)O gave amounts of 18O in the N2O product consistent with equilibration of nitric oxide with a small pool of free nitrite . Using 15NO and NH2OH, competition between denitrification and nitrosation reactions was demonstrated, as is required if the enzyme-nitrosyl complex is an intermediate in both nitrosation and denitrification reactions . The first evidence for exchange of 18O between H2(18)O and a nitrosation intermediate occurring after the enzyme-nitrosyl complex, presumably an enzyme-bound nitrosamine, has been obtained . The collective results are most consistent with denitrification N2O originating via attack of NO-2 on a coordinated nitrosyl, as proposed earlier (Averill, B . A., and Tiedje, J . M . (1982) FEBS Lett . 138, 8-11). Eur J Biochem, 1986 Jul 15, 158(2), 429 - 36 The respiratory nitrate reductase from Paracoccus denitrificans . Molecular characterisation and kinetic properties; Craske A et al.; The respiratory nitrate reductase from Paracoccus denitrificans has been purified in the non-ionic detergent Nonidet P-40 . The enzyme comprises three polypeptides, alpha, beta and gamma with estimated relative molecular masses of 127 000, 61 000 and 21 000 . Duroquinol or reduced-viologen compounds acted as the reducing substrates . The nitrate reductase contained a b-type cytochrome that was reduced by duroquinol and oxidised by nitrate . A preparation of the enzyme that lacked both detectable b-type cytochrome and the gamma subunit was obtained from a trailing peak of nitrate reductase activity collected from a gel filtration column . Absence of the gamma subunit correlated with failure to use duroquinol as reductant; activity with reduced viologens was retained . It is concluded that in the plasma membrane of P . denitrificans the gamma subunit catalyses electron transfer to the alpha and beta subunits of nitrate reductase from ubiquinol which acts as a branch point in the respiratory chain . A new assay was introduced for both nitrate and quinol-nitrate oxidoreductase activity . Diaphorase was used to couple the oxidation of NADH to the production of duroquinol which acted as electron donor to nitrate reductase . Under anaerobic conditions absorbance changes at 340 nm were sensitive to nitrate concentrations in the low micromolar range . This coupled assay was used to determine that the purified enzyme had Km(NO-3) of 13 microM and a Km of 470 microM for ClO-3, an alternative substrate . With viologen substrates Km(NO-3) of 283 microM and Km(ClO-3) of 470 microM were determined; the enzymes possessed a considerably higher Vmax with either NO-3 or ClO-3 than was found when duroquinol was substrate . Azide was a competitive inhibitor of nitrate reduction in either assay system (Ki = 0.55 microM) but 2-n-heptyl-4-hydroxyquinoline N-oxide was effective only with the complete three-subunit enzyme and duroquinol as substrate, consistent with a site of action for this inhibitor on the b-type cytochrome . The low Km for nitrate observed in the duriquinol assay is comparable with the apparent Km(NO-3) recently reported for intact cells of P . denitrificans {Parsonage, D., Greenfield, A . J . & Ferguson, S . J . (1985) Biochim . Biophys . Acta 807, 81-95} . This similarity is discussed in terms of a possible requirement for a nitrate transport system . The nitrate reductase system from P . denitrificans is compared with that from Escherichia coli. J Biol Chem, 1986 Jul 5, 261(19), 8577 - 80 Characterization of two inducible periplasmic c-type cytochromes from Paracoccus denitrificans; Husain M et al.; When grown on methanol or methylamine, Paracoccus denitrificans synthesized three periplasmic soluble c-type cytochromes, cytochrome c550 and two additional cytochromes which were not present during growth on succinate and have not previously been characterized . These cytochromes have been separated from each other and their physical properties have been determined . The inducible cytochromes, c551i and c553i, exhibit Mr and pI values of 22,000 and 3.5, and 30,000 and 3.8, respectively . Cytochrome c553i binds CO . None of these cytochromes accepted electrons directly from methylamine dehydrogenase, but each accepted electrons from amicyanin, the electron acceptor for methylamine dehydrogenase . Cytochrome c551i was the most efficient electron acceptor for amicyanin, exhibiting a half-time of reaction at least 6 and 15 times faster than those observed for cytochromes c553i and c550, respectively. Can J Microbiol, 1986 Jul, 32(7), 543 - 7 Temporary low oxygen conditions for the formation of nitrate reductase and nitrous oxide reductase by denitrifying Pseudomonas sp . G59; Aida T et al.; Formation of nitrate reductase (NaR) and nitrous oxide reductase (N2OR) by a Pseudomonas sp . G59 did not occur in aerobic or anaerobic conditions, but was observed in a microaerobic incubation in which an anaerobically grown culture was agitated in a sealed vessel initially containing 20 kPa oxygen in the headspace . During the microaerobic incubation, the oxygen concentration in the headspace decreased and dissolved oxygen reached 0.1-0.2 kPa . NaR activity was detected immediately and N2OR activity after 3 h of incubation irrespective of the presence or absence of NO3- or N2O . In the presence of NO3-, NO2- was accumulated as a major product, but N2O was observed in low concentrations only after N2OR appeared . After microaerobic incubation for 3 h, N2OR formation continued even anaerobically in an atmosphere of N2O . In contrast, Escherichia coli formed NaR not only microaerobically but also anaerobically . However, NaR formation by E . coli was inhibited by sodium fluoride under anaerobic, but not under microaerobic conditions . The Pseudomonas culture did not possess fermentative activity . It is suggested that the dependence on microaerobiosis for the formation of these reductases by the Pseudomonas culture was due to an inability to produce energy anaerobically until these anaerobic respiratory enzymes were formed. J Bacteriol, 1986 Jun, 166(3), 812 - 7 Electron transfer flavoprotein from Methylophilus methylotrophus: properties, comparison with other electron transfer flavoproteins, and regulation of expression by carbon source; Davidson VL et al.; When grown on methylated amines as a carbon source, Methylophilus methylotrophus synthesizes an electron transfer flavoprotein (ETF) which is the natural electron acceptor of trimethylamine dehydrogenase . It is composed of two dissimilar subunits of 38,000 and 42,000 daltons and 1 mol of flavin adenine dinucleotide . It was reduced by trimethylamine dehydrogenase to a stable anionic semiquinone form, which could not be converted, either enzymatically or chemically, to the fully reduced dihydroquinone . This ETF exhibited spectral properties which were nearly identical to ETFs from bacterium W3A1, Paracoccus denitrificans, and pig liver mitochondria . M . methylotrophus ETF cross-reacted immunologically and enzymatically with the ETF of bacterium W3A1 but not with the other two ETFs . In M . methylotrophus and bacterium W3A1, ETF and trimethylamine dehydrogenase were each expressed during growth on trimethylamine and were each absent during growth on methanol. Biochemistry, 1986 May 6, 25(9), 2431 - 6 Properties of Paracoccus denitrificans amicyanin; Husain M et al.; Paracoccus denitrificans synthesizes an inducible, periplasmic, blue copper protein {Husain, M., & Davidson, V.L . (1985) J . Biol . Chem . 260, 14626-14629} that can be classified as an amicyanin on the basis of its ability to accept electrons from methylamine dehydrogenase . The amino acid composition and sequence of the 10 N-terminal residues of this protein have been determined . From these data, it is evident that amicyanin is structurally distinct from azurins as it contains no disulfide bond and an N-terminal sequence that is completely different from the highly conserved N-terminal azurin sequences . Dialysis of reduced amicyanin against potassium cyanide resulted in a nearly quantitative yield of apoamicyanin . Amicyanin and apoamicyanin exhibit fluorescence emission maxima at 314 nm when excited at 280 nm . Addition of 6 M guanidine hydrochloride shifts these emission maxima to 350 nm . The fluorescence intensity of apoamicyanin is 10-fold greater than that of amicyanin . Addition of copper to the apoprotein caused a stoichiometric quenching of fluorescence and restoration of visible absorbance with no concomitant change in absorbance at 280 nm . At least one cysteine residue, which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) in apoamicyanin, does not react in the holoprotein, even in the presence of 6 M guanidine hydrochloride . Reductive and oxidative titrations of amicyanin indicate that it is a one-electron carrier . This amicyanin is also able to accept electrons from the methylamine dehydrogenase isolated from bacterium W3A1, which is taxonomically very different from P . denitrificans. J Mol Biol, 1986 May 5, 189(1), 257 - 8 Preliminary X-ray crystallographic study of amicyanin from Paracoccus denitrificans; Lim LW et al.; Single crystals have been prepared of Paracoccus denitrificans amicyanin, a blue copper protein that serves as an electron acceptor for methylamine dehydrogenase . The crystals belong to the monoclinic space group P2(1), and have unit cell parameters a = 20.90 A, b = 56.61 A, c = 27.55 A and beta = 96.41 . There is one molecule in the asymmetric unit . The crystals diffract to beyond 1.5 A resolution. J Bacteriol, 1986 May, 166(2), 604 - 8 Properties and electron transfer specificity of copper proteins from the denitrifier "Achromobacter cycloclastes"; Liu MY et al.; A blue copper protein (Mr 12,000) was purified from cells of "Achromobacter cycloclastes" grown as a denitrifier . When reduced, the blue copper protein transferred electrons to the copper protein nitrite reductase purified from the same cells, whereas a variety of cytochromes from denitrifiers failed to do so . Inclusion of a protease inhibitor, phenylmethylsulfonyl fluoride, in the buffers employed during preparation yielded purified blue copper protein with 18 more amino acid residues and two times more specific enzyme activity than other researchers have found. Anal Biochem, 1986 May 1, 154(2), 470 - 5 Multiple-phase equilibration headspace analysis for the determination of N2O and N2 during bacterial denitrification; Byrne MD et al.; A gas-handling manifold for the preparation, introduction and analysis by gas chromatography (GC) system of the gaseous products of denitrification is described . A procedure of multiple-phase equilibration is adopted which allows the quantitative determination of the total gas present in sample vials . Assumptions of solubility coefficients are not required as these are determined during the analysis . The method is particularly suited to gases of appreciable solubilities as a significant proportion of the gas will be found in the liquid phase . This method was used for