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T C Hollocher

Publications and source records attributed to T C Hollocher.

At least 19 recordsLinked to original sources
PubMed1993

A diffusion-controlled step in the catalytic cycle of nitrous oxide reductase from Wolinella succinogenes.

The viscosity test was applied to the highly active nitrous oxide reductase purified from Wolinella succinogenes to determine whether the catalytic cycle may contain a diffusion-controlled step. For this test, the benzyl viologen cation (BV+)-N2O oxidoreductase reaction, which exhibits a kcatBV/KmBV approximately 2 x 10(8) M-1 s-1 at 23 degrees C and pH 7.1, was run in solutions of nominally nondenaturing viscogens that were used to increase microviscosity. The parameters kcatN2O and kcatBV were unaffected by viscosity in the range of viscogen concentrations that were not inhibitory and where the data were well behaved. KmBV, but not KmN2O, was observed to increase linearly with relative viscosity with a slope of 0.14 for all viscogens surveyed. The results, when considered in the context of a plausible kinetic model for the BV(+)-N2O oxidoreductase reaction, suggest that one of the two one-electron reactions between BV+ and enzyme is diffusion-controlled but only partially rate determining. The ratio of the second-order-rate constants for these two one-electron steps is estimated to be about 6.1, and the larger rate constant to be about 1.1 x 10(9) M-1 s-1. There would appear to be no diffusion-controlled step associated with the half-reaction which results in reduction of N2O to N2.

Diffusion↗
PubMed1992

An apparently allosteric effect involving N2O with the nitrous oxide reductase from Wolinella succinogenes.

It was shown that kcat for the benzyl viologen cation (BV+)-N2O oxidoreductase activity of nitrous oxide reductase from Wolinella succinogenes was 2-3 times greater at high N2O concentrations than at low. This effect of N2O on kcat exhibited a titration curve implicating a single secondary binding site for N2O with a Kd of 130-200 microM (Km with respect to N2O is about 2.5 microM). This work represents the first evidence of an apparently allosteric kinetic effect among nitrous oxide reductases. Its possible cause is discussed. BV+ was generated in these kinetic studies by addition of sub-stoichiometric amounts of dithionite. This means of reduction proved to be superior to the photochemical generation of BV+ that had been used previously with the enzyme. Mass spectrometric measurements suggested that the M(r) of the subunit of the enzyme is about 95,500 rather than 88,000.

Allosteric Regulation↗
PubMed1992

Oxidation of dithiothreitol during turnover of nitric oxide reductase: evidence for generation of nitroxyl with the enzyme from Paracoccus denitrificans.

The stoichiometric relationship between thiol oxidized and NO reduced was studied for the reaction catalyzed by nitric oxide reductase from Paracoccus denitrificans. The reaction systems consisted of dithiothreitol, ascorbate, phenazine methosulfate, enzyme and NO, or that system minus ascorbate. The mole ratio of thiol groups oxidized to NO reduced was observed to be 2.3 to 1.5 over a range of NO from 0.09 to 0.35 mumol. A ratio of 1.0 was expected for the simple reduction of NO by 1-electron to N2O. The oxidation of additional thiol is attributed to the trapping of nitrosyl hydride (nitroxyl, NO/NOH) by thiol.

Dithiothreitol↗
PubMed1991

Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characteristics.

Nitric oxide reductase was purified from Paracoccus denitrificans very nearly to homogeneity by a simple method that involved the use of octyl glucoside to solubilize the enzyme from membranes and required a single hydroxyapatite column. The enzyme had specific activities of about 10 mumol NO reduced x min-1 x mg-1 at pH 6.5 in an amperometric assay system using phenazine methosulfate/ascorbate as the reducing agent and about 22 mumol NO reduced x min-1 x mg-1 at pH 5.0, which is the optimum pH. These values are based on average rates over kinetically complex progress curves and would be about three times greater if based on maximum rate values. The enzyme appeared to be reversibly inhibited by NOaq and to have a Km too low (probably less than or equal to 1 microM) to measure reliably by the amperometric method. The effective second-order rate constant of the enzyme lay within 1 to 2 orders of magnitude of the diffusion controlled limit. The enzyme was composed of a tight complex of two cytochromes: a cytochrome c (Mr = 17,500) and a cytochrome b (Mr = 38,000). The mole ratios of cytochrome c to cytochrome b and Mr 17,500 peptide to Mr 38,000 peptide were both about 1.7, and the heme content was about 3 mol/73,000 g (38,000 + 2(17,500)). Each subunit therefore contained only one heme group. The Mr 38,000 peptide aggregated when heated in the sample buffer used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the ascorbate-based activity, the enzyme showed a little NADH-NO oxidoreductase activity which was not inhibited by antimycin A. The enzyme lost activity with a half-life of about 2 days at 4 degrees C but could be preserved at -20 degrees C and in liquid nitrogen. It seemed not to be inactivated by aerobic solutions. These observations, and the recent ones by Carr and Ferguson (Carr, G.J., and Ferguson, S.J. (1990) Biochem. J. 269, 423-429) with a partially purified preparation of nitric oxide reductase, establish that the enzyme from Pa. denitrificans is a cytochrome bc complex which resembles that from Pseudomonas stutzeri (Heiss, B., Frunzke, K., and Zumft, W.G. (1989) J. Bacteriol. 171, 3288-3297). There would appear to be no functional relationship between nitric oxide reductase and a Mr = 34,000 peptide of Pa. denitrificans membranes reported previously to be present in purified preparations of a nitric oxide reductase (Hoglen, J., and Hollocher, T.C. (1989) J. Biol. Chem. 264, 7556-7563).

Amino Acids↗
PubMed1991

Catalysis of nitrosyl transfer by denitrifying bacteria is facilitated by nitric oxide.

Two denitrifying bacteria, Pseudomonas stutzeri and Achromobacter cycloclastes, were incubated with Na15NO2 and NaN3 under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide. This transfer, which is presumed to be mediated by the heme- and copper-containing nitrite reductase of P. stutzeri and A. cycloclastes, respectively, leads to formation of isotopically mixed 14,15N2O, whereas denitrification leads to 15N2O. The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of 15NO. Absorption of NO from the gas phase by acidic CrSO4 in a sidewell largely abolished nitrosyl transfer to azide. With these two organisms, which are thought to be representative of denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO.

Alcaligenes↗
PubMed1991

Electron paramagnetic resonance observations on the cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes.

Nitrous oxide reductase from Wolinella succinogenes, an enzyme containing one heme c and four Cu atoms/subunit of Mr = 88,000, was studied by electron paramagnetic resonance (EPR) at 9.2 GHz from 6 to 80 K. In the oxidized state, low spin ferric cytochrome c was observed with gz = 3.10 and an axial Cu resonance was observed with g parallel = 2.17 and g perpendicular = 2.035. No signals were detected at g values greater than 3.10. For the Cu resonance, six hyperfine lines each were observed in the g parallel and g perpendicular regions with average separations of 45.2 and 26.2 gauss, respectively. The hyperfine components are attributed to Cu(I)-Cu(II) S = 1/2 (half-met) centers. Reduction of the enzyme with dithionite caused signals attributable to heme c and Cu to disappear; exposure of that sample to N2O for a few min caused the reappearance of the g = 3.10 component and a new Cu signal with g parallel = 2.17 and g perpendicular = 2.055 that lacked the simple hyperfine components attributed to a single species of half-met center. The enzyme lost no activity as the result of this cycle of reduction and reoxidation. EPR provided no evidence for a Cu-heme interaction. The EPR detectable Cu in the oxidized and reoxidized forms of the enzyme comprised about 23 and 20% of the total Cu, respectively, or about one spin/subunit. The enzyme offers the first example of a nitrous oxide reductase which can have two states of high activity that present very different EPR spectra of Cu. These two states may represent enzyme in two different stages of the catalytic cycle.

Bacteroidaceae↗
PubMed1991

Extended x-ray absorption fine structure and electron paramagnetic resonance of nitrous oxide reductase from Pseudomonas aeruginosa strain P2.

The copper centers of nitrous oxide reductase from Pseudomonas aeruginosa strain P2 were studied by x-ray and electron paramagnetic resonance (EPR) spectroscopy. The enzyme is dimeric and contains four Cu atoms and about seven cysteine residues/subunit of Mr = 73,000. The extended x-ray absorption fine structure (EX-AFS) spectrum was analyzed for enzyme as isolated (oxidized or slightly reduced), enzyme exposed briefly to air, reduced enzyme, and enzyme at pH 7 after having been activated by standing at pH 10. The average Cu ligand environment in the first shell was best modeled for all forms of the enzyme by a combination of N/O and S atoms at a total coordination number between 3 and 4 and bond distances ranging from 1.96-2.03 A for Cu-N/O and 2.20-2.25 A for Cu-S. The data could be fit without using Cu-Cu interactions. Overall the results are similar to those reported for the enzyme for Pseudomonas stutzeri (Scott, R. A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086). The first derivative EPR spectra of the Cu(II) centers at 15 and 45 K were qualitatively similar among enzyme as isolated and enzyme exposed to N2O or air. These three nominally oxidized samples showed an axial signal with g perpendicular = 2.03 and g parallel = 2.15-2.16. Hyperfine structure was observed in both the g parallel and g perpendicular regions with splittings of 43 and 25 gauss, respectively. These hyperfine components are attributed to exchange coupled Cu(I)-Cu(II) S = 1/2 (half-met) centers. In the enzyme as isolated and after exposure to N2O, about 3/4 of the Cu was EPR silent, whereas after exposure to air the signal integrated to about half the Cu concentration. The EPR spectrum of enzyme activated at pH 10 but frozen at pH 7 was a composite of spectra from activated and inactive species. The activated species presented a complex set of narrow hyperfine components which may arise from contributions from more than one species of half-met center.

Copper↗
PubMed1991

Purification and characterization of nitrous oxide reductase from Pseudomonas aeruginosa strain P2.

Nitrous oxide reductase, which catalyzes the reduction of N2O to N2, was purified in a largely oxidized form from Pseudomonas aeruginosa strain P2 by a simple anaerobic procedure to yield an enzyme with a peptide purity of 95-98%. For the native (dimeric) enzyme, Mr = 120,000 and for the denatured subunit, Mr = 73,000. The enzyme contained four Cu atoms/subunit, was purple in color, and exhibited a broad absorption band at 550 nm with an extinction coefficient of about 11,000 M-1 x cm-1 referenced to the dimer. It was nearly inactive as prepared but could be activated by incubation with 2-(N-cyclohexylamino)ethane sulfonate buffer, pH 10, to specific activities as high as 27 mumol of N2O x min-1 x mg-1.Km for N2O and benzyl viologen radical cation was about 2 and 4 microM, respectively, both before and after enzyme activation. Activation increased the t1/2 for turnover-dependent inactivation from about 30 s to 5-10 min. Reduction of the enzyme by dithionite was kinetically biphasic and resulted in the loss of the 550-nm band and ultimate appearance of a 670-nm band. Isoelectric focusing revealed five components with pI values from 5.2 to 5.7. The pI values did not change following activation. The copper CD spectrum of the enzyme as prepared was different from that for the activated enzyme, whereas those for the enzyme after exposure to air and the activated enzyme were similar. Because the activated enzyme is a mixture of activated and inactive species, the specific activity of the activated species must be substantially greater than the observed value. Molecular heterogeneity may also explain the decreased optical absorbance and CD amplitude that resulted from the activation process. The data overall reinforce the view that the absorption spectrum of nitrous oxide reductase is not a good predictor of absolute activity.

Amino Acids↗
PubMed1990

Steady-state nitric oxide concentrations during denitrification.

Three species of denitrifying bacteria, Paracoccus denitrificans, Pseudomonas stutzeri strain JM300, and Achromobacter cycloclastes, were allowed to reduce nitrate or nitrite in anaerobic, closed vials while the equilibration of gases between aqueous and gas phases was facilitated by vigorous stirring. The gas phase was sampled and analyzed for NO with use of a chemiluminescence detector calibrated against bottled NO standards or against NO produced by the nitrite-iodide reaction. [NOaq] was inferred from [NOg] and the solubility of NO. NO was detected only during denitrification in amounts that, once established, did not change with time, were independent of the initial concentration of nitrate or nitrite, and were largely independent of cell concentration, at least when nitrate was the oxidant. The usual level of NO was promptly re-established following the addition of exogenous NO or following the loss of NO by sparging. The aforementioned properties are expected for a steady-state intermediate in denitrification. Steady-state [NOaq] ranged between 1 and 65 nM depending on species and conditions. Similar results were also obtained in a related experiment in which P. stutzeri strain ZoBell respired nitrite under growth conditions. The very low steady-state [NOaq] observed during denitrification imply that the maximum activity of nitric oxide reductase in vivo, if it could be realized, would be large relative to that for nitrite reductase. This circumstance allows NO to be an intermediate without reaching toxic steady-state levels.

Alcaligenes↗
PubMed1990

The kinetic and isotopic competence of nitric oxide as an intermediate in denitrification.

Rates of NO uptake by five denitrifying bacteria were estimated by NO-electrode and gas chromatography methods under conditions of rather low cell densities and [NOaq]. The rates so measured, VmaxNO, represent lower limits for the true value of that parameter, but nevertheless exceed Vmax for nitrite uptake, VmaxNi, by a factor of two typically. Previous estimates under suboptimal conditions had placed VmaxNO at 0.3-0.5 of VmaxNi (St. John, R. T., and Hollocher, T. C. (1977) J. Biol. Chem. 252, 212-218; Garber, E. A. E., and Hollocher, T.C. (1981) J. Biol. Chem. 256, 5459-5465). The steady-state [NOaq] during denitrification of nitrite by nitrate-grown cells was less than or equal to 1 microM. The above observations, taken with a recent direct estimate for the KmNO for NO uptake of 0.4 microM (Zafiriou, O. C., Hanley, Q. S., and Snyder, G. (1989) J. Biol. Chem. 264, 5694-5699), would allow NO to be a free intermediate between nitrite and N2O with steady-state concentrations of less than or equal to 0.4 microM. As the result of special conditions during cell growth or differential inhibition by azide, it was possible to establish systems that accumulated NO during denitrification of nitrite. In all such cases, VmaxNO less than VmaxNi, and the time required to reach the maximum [NOaq] corresponded closely to the time needed to exhaust the nitrite. A semiquantitative isotope experiment with Paracoccus denitrificans demonstrated the trapping of 15NO from 15NO2- in a pool of NOaq. A quantitative isotope method using low densities of the same bacterium showed that 15N from 15NO2- and 14N from NOg combine randomly to form N2O during the simultaneous denitrification of 15NO2- and NO. The result requires that the pathways from nitrite and NO share a common mononitrogen intermediate. Results to the contrary obtained at high cell densities (first two references cited above) are now believed to have been due to technical artifacts. The present results are consistent with the view that NO is under kinetic control as a free intermediate in denitrification and serve to remove previously imagined constraints on this view.

Bacteria↗
PubMed1989

Purification and some characteristics of nitric oxide reductase-containing vesicles from Paracoccus denitrificans.

Nitric oxide reductase of Paracoccus denitrificans was purified, with the use of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) detergent, as membrane vesicles of apparent Mr = 2-3 x 10(6). Fifty percent of the protein was a peptide of Mr = 34,000. Further fractionation with sodium dodecyl sulfate (SDS) resulted in vesicles in which the peptide constituted 90-95% of the protein. This peptide, which is rich in Ala, Gly, Ser, Asx, and Glx, is considered to be the peptide of nitric oxide reductase. The CHAPSO- and SDS-fractionated preparations lost activity at 4 degrees C, pH 7.4, with half-times, respectively, of about 6 days and 4 h. Specific activities at 32 degrees C, pH 7.4, of about 0.33 mumol of NO x min-1 x mg-1 were realized after fractionation with CHAPSO in a phenazine methosulfate/ascorbate-based assay. The Km(NO) was less than or equal to 17 microM at pH 7.4. Rates decreased substantially below pH 5 and above pH 7.6. The preparations were free or almost free of cytochromes, exhibited otherwise no absorption bands in the visible region, contained no redox metals except for very small amounts of iron, were not inhibited by EDTA or some other common inhibitors of redox-metal enzymes, and were not observed to catalyze the reduction of nitrate, nitrite, or N2O. An absorption band at 274 nm in both the CHAPSO- and SDS-fractionated preparations was attributed to the presence of a solvent-soluble chromophore. N-Bromosuccinimide (NBS) inactivated the enzyme and bleached the chromophore both in the enzyme preparation and, after its purification, in 95% ethanol. NBS-inactivated enzyme could be reconstituted with purified chromophore, which alone seemed to have no nitric oxide reductase activity, but not with purified chromophore that had been reacted with NBS. Spectral changes interpretable as due to changes in redox state were not observed when enzyme was exposed to NO or certain reducing agents.

Amino Acids↗
PubMed1989

Purification and some characteristics of a cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes.

Nitrous oxide reductase from Wolinella succinogenes was purified very nearly to homogeneity. The enzyme was found to be dimeric, with Mr = 162,000 and subunit Mr = 88,000, and to contain three copper atoms and one iron atom (as cytochrome c) per subunit. The oxidized enzyme exhibited absorption bands at 410 and 528 nm, and the dithionite-reduced enzyme, at 416, 520, and 550 nm. The isoelectric point was 8.6; specific activity was at 25 degrees C and pH 7.1, 160 mumol x min-1 x mg-1; and Km was 7.5 microM N2O under the same conditions. alpha-Chymotrypsin cleaved the enzyme into cytochrome c-depleted dimers with an average Mr = 134,000 and cytochrome c-enriched fragments with an average Mr = 13,000. The enzyme was stable at 4 degrees C for at least 100 h under air and 3 h in the presence of 5 mM EDTA. It exhibited a dithionite-N2O oxidoreductase as well as a BV+-N2O oxidoreductase activity. During turnover with BV+ at 25 mM N2O, the enzyme was observed to undergo an initial activation and a subsequent inactivation. The kinetics of inactivation were approximately first-order in remaining activity, and the first-order rate constant was essentially independent of the initial enzyme concentration. These characteristics are consistent with the occurrence of turnover-dependent inactivation. Acetylene was a relatively weak inhibitor, but cyanide and azide were rather strong inhibitors. The nitrous oxide reductase of W. succinogenes is quite different from that of denitrifying bacteria. The amount of activity in cell extracts and the absence of O2-labile nitrous oxide reductase suggested that the cytochrome c containing enzyme may be the only one produced by W. succinogenes.

Bacteroidaceae↗
PubMed1989

Some characteristics of a chromophoric lipid associated with nitric oxide reductase from Paracoccus denitrificans.

A lipid with a UV chromophore (lambda max = 274 nm) was purified in small amounts from nitric oxide reductase of Paracoccus denitrificans and determined to have a molecular weight of 686, a most probable formula of C43H78O4N2 and about two phenylhydrazine-reactive carbonyls. On the basis of 1H and 13C-NMR, IR and mass spectrometric studies, the chromophoric lipid was inferred to have something like bilateral symmetry and to contain ketone-like carbonyls, alkene centers and at least three alkyl or olefinic chains. Several likely substructures are discussed and an enaminoketone is considered as a model for the chromophore. Evidence was lacking for phosphate, an aromatic ring, a 1,4-quinone, polyunsaturated fatty acyl groups, isoprenyl/isopranyl chains and simple alkyl acids, esters, amides and ethers. The compound appears to have some novel features. On the basis of reconstitution studies, the chromophoric lipid may be essential for nitric oxide reductase activity.

Chemical Phenomena↗
PubMed1988

Reduction of nitrite to nitric oxide by enteric bacteria.

Seven bacteria representing seven genera of enteric bacteria, in addition to Escherichia coli, were shown to reduce nitrite to NO under anaerobic conditions when the cells were grown as nitrate respirers. NO production was inhibited by nitrate and azide and was self limiting, just as was found to be the case previously with E. coli and its nitrate reductase. Maximum initial rates of NO production were observed at pH 5.5-6.

Enterobacteriaceae↗
PubMed1988

Trapping of nitric oxide produced during denitrification by extracellular hemoglobin.

A spectrophotometric method has been developed that uses extracellular hemoglobin (Hb) to trap nitric oxide (NO) released during denitrification as nitrosyl hemoglobin (HbNO). The rate of complexation of NO with Hb is about at the diffusion controlled limit for protein molecules and the product, HbNO, is essentially stable. Hb was added to an anaerobic bacterial suspension and denitrification was initiated with either KNO2 or KNO3. HbNO formation was observed for six species of denitrifying bacteria and showed isosbestic points at 544, 568, and 586 nm. Cellular NO production, presumably by nitrite reductase, was kinetically distinct from the much slower chemical reaction of Hb with KNO2 to form methemoglobin and HbNO. The rate of HbNO formation was proportional to cell density, essentially independent of pH from 6.8 to 7.4, nearly zero order in [Hb] and, at least with Paracoccus denitrificans, strongly inhibited by rotenone and antimycin A. The Cu chelator, diethyldithiocarbamate, had no effect on HbNO formation by Pa. denitrificans, but abolished that by Achromobacter cycloclastes which uses a Cu-containing nitrite reductase known to be inactivated by the chelator. HbNO formation did not occur with non-denitrifying bacteria. The stoichiometry at high [Hb] for conversion of Hb to HbNO was 1.3-1.8 KNO2 per Hb for Pa. denitrificans, Pseudomonas aeruginosa, and A. cycloclastes and about 3.4 for Pseudomonas stutzeri. The former range of values corresponds to a partition of about 2 N atoms in 3 toward trapping and 1 in 3 toward reduction on the pathway to N2. Nitrogen not trapped appeared largely as N2O in presence of acetylene. The results are consistent with a model in which NO is a freely diffusible intermediate between nitrite and N2O, providing that nitric oxide reductase is or nearly is a diffusion controlled enzyme.

Antimycin A↗
PubMed1988

Mechanism for nitrosation of 2,3-diaminonaphthalene by Escherichia coli: enzymatic production of NO followed by O2-dependent chemical nitrosation.

The mechanism by which Escherichia coli can catalyze the nitrite-dependent nitrosation of 2,3-diaminonaphthalene (DAN), with formation of the corresponding fluorescent triazole, was studied. The reaction was dependent on production of a gaseous compound which can nitrosylate DAN upon contact with air. This compound was identified as nitric oxide (NO), and the kinetics of NO and triazole production are reported. NO and triazole were produced proportionally in a stoichiometric ratio, NO/triazole, of 1.4 to 1.7. Given the requirement for air, nitrosation of DAN probably proceeds via formation of the well-known strong nitrosylating agents N2O3 and N2O4 from NO. The parallel inhibition of NO and triazole production by azide and nitrate served to reinforce the link between nitrosation and nitrate reductase that had been established previously by others on genetic grounds.

2-Naphthylamine↗