Biomedical subjects

D J Arp

Publications and source records attributed to D J Arp.

At least 55 records · Page 3Linked to original sources

PubMed1993

An electrophoretic study of the thermal- and reductant-dependent aggregation of the 27 kDa component of ammonia monooxygenase from Nitrosomonas europaea.

Standard protocols for sample preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) typically involve the combined use of heat and a reductant to fully disrupt protein-protein interactions and allow for constant ratios of SDS-binding to individual polypeptides. However, 14C-labeled forms of the membrane-bound, active-site-containing 27 kDa polypeptide of ammonia monooxygenase from Nitrosomonas europaea undergo an aggregation reaction when cells or membranes are heated in the presence of SDS-PAGE sample buffer. The aggregate produced after heating at 100 degrees C is a soluble complex which fails to enter the stacking gel in discontinuous SDS-PAGE gels. The extent of the aggregation reaction is dependent on the temperature of sample preparation, and the reaction exhibits first-order kinetics at 65 degrees C and 100 degrees C (rates constants = 0.07 and 0.35 min-1, respectively). The rate of the aggregation reaction is further dependent on the concentration of reductant used in the sample buffer. However, the concentration of SDS does not significantly affect the rate of aggregation. The aggregated form of the 27 kDA polypeptide can be isolated by gel-permeation chromatography in the presence of SDS. The aggregated protein can also be returned to the monomeric state by incubation at high pH in the presence of SDS. The aggregation reaction also occurs with 14C2H2-labeled polypeptides in other species of autotrophic nitrifiers and a methanotrophic bacterium which expresses the particulate form of methane monooxygenase. We conclude that strongly hydrophobic amino acid sequences present in ammonia monooxygenase are responsible for the aggregation phenomenon.

Carbon Dioxide↗

PubMed1993

Inhibition of Ammonia Oxidation in Nitrosomonas europaea by Sulfur Compounds: Thioethers Are Oxidized to Sulfoxides by Ammonia Monooxygenase.

Organic sulfur compounds are well-known nitrification inhibitors. The inhibitory effects of dimethylsulfide, dimethyldisulfide, and ethanethiol on ammonia oxidation by Nitrosomonas europaea were examined. Both dimethylsulfide and dimethyldisulfide were weak inhibitors of ammonia oxidation and exhibited inhibitory characteristics typical of substrates for ammonia monooxygenase (AMO). Depletion of dimethylsulfide required O(2) and was prevented with either acetylene or allylthiourea, two inhibitors of AMO. The inhibition of ammonia oxidation by dimethylsulfide was examined in detail. Cell suspensions incubated in the presence of ammonia oxidized dimethylsulfide to dimethyl sulfoxide. Depletion of six other thioethers was also prevented by treating cell suspensions with either allylthiourea or acetylene. The oxidative products of three thioethers were identified as the corresponding sulfoxides. The amount of sulfoxide formed accounted for a majority of the amount of sulfide depleted. By using gas chromatography coupled with mass spectrometry, allylmethylsulfide was shown to be oxidized to allylmethylsulfoxide by N. europaea with the incorporation of a single atom of O derived from O(2) into the sulfide. This result supported our conclusion that a monooxygenase was involved in the oxidation of allylmethylsulfide. The thioethers are concluded to be a new class of substrates for AMO. This is the first report of the oxidation of the sulfur atom by AMO in whole cells of N. europaea. The ability of N. europaea to oxidize dimethylsulfide is not unique among the ammonia-oxidizing bacteria. Nitrosococcus oceanus, a marine nitrifier, was also demonstrated to oxidize dimethylsulfide to dimethyl sulfoxide.

Journal Article↗

PubMed1993

Mechanism-Based Inactivation of Ammonia Monooxygenase in Nitrosomonas europaea by Allylsulfide.

Allylsulfide caused an irreversible inactivation of ammonia monooxygenase (AMO) activity (ammonia-dependent O(2) uptake) in Nitrosomonas europaea. The hydroxylamine oxidoreductase activity (hydrazine-dependent O(2) uptake) of cells was unaffected by allylsulfide. Anaerobic conditions or the presence of allylthiourea, a reversible noncompetitive AMO inhibitor, protected AMO from inactivation by allylsulfide. Ammonia did not protect AMO from inactivation by allylsulfide but instead increased the rate of inactivation. The inactivation of AMO followed pseudo-first-order kinetics, but the observed rates did not saturate with increasing allylsulfide concentrations. The time course of recovery of AMO-dependent nitrite production after complete inactivation by allylsulfide required de novo protein synthesis. Incubation of cells with allylsulfide prevented the C label from C(2)H(2) (a suicide mechanism-based inactivator of AMO) from being incorporated into the 27-kDa polypeptide of AMO. Some compounds structurally related to allylsulfide were unable to inactivate AMO. We conclude that allylsulfide is a specific, mechanism-based inactivator of AMO in N. europaea.

Journal Article↗

PubMed1993

Kinetic Studies of Ammonia Monooxygenase Inhibition in Nitrosomonas europaea by Hydrocarbons and Halogenated Hydrocarbons in an Optimized Whole-Cell Assay.

The inhibitory effects of 15 hydrocarbons and halogenated hydrocarbons on NH(3) oxidation by ammonia monooxygenase (AMO) in intact cells of the nitrifying bacterium Nitrosomonas europaea were determined. Determination of AMO activity, measured as NO(2) production, required coupling of hydroxylamine oxidoreductase (HAO) activity with NH(3)-dependent NH(2)OH production by AMO. Hydrazine, an alternate substrate for HAO, was added to the reaction mixtures as a source of reductant for AMO. Most inhibitors exhibited competitive or noncompetitive inhibition patterns. The competitive character generally decreased (K(i) (E)/K(i) (ES) increased) as the molecular size of the inhibitors increased. For example, CH(4) and C(2)H(4) were competitive inhibitors of NH(3) oxidation, whereas the remaining alkanes (up to C(4)) and monohalogenated (Cl, Br, I) alkanes were noncompetitive. Oxidation of C(2)H(5)Br (noncompetitive) increased as the NH(4) concentration increased up to 40 mM, whereas oxidations of inhibitors with competitive character (K(i) (E) << K(i) (ES)) were diminished at 40 mM NH(4). Multichlorinated compounds produced nonlinear Lineweaver-Burk plots. Iodinated alkanes (CH(3)I, C(2)H(5)I) and C(2)Cl(4) were potent inhibitors of NH(3) oxidation. Maximum rates of NH(3), C(2)H(4), and C(2)H(6) oxidations were approximately equivalent, suggesting a common rate-determining step. These data support an active-site model for AMO consisting of an NH(3)-binding site and a second site that binds noncompetitive inhibitors, with oxidation occurring at either site.

Journal Article↗

PubMed1993

In Azotobacter vinelandii hydrogenase, substitution of serine for the cysteine residues at positions 62, 65, 294, and 297 in the small (HoxK) subunit affects H2 oxidation [corrected].

The essential role of the small (HoxK) subunit of hydrogenase of Azotobacter vinelandii in H2 oxidation was established. This was achieved by modification of the two Cys-X2-Cys amino acid motifs at the N and C termini of the HoxK subunit (Cys-62, -65, -294, and -297). The Cys codons were individually mutated to Ser codons. Modifications in these two motifs resulted in loss of hydrogenase activity. At the N terminus, the mutations of the codons for the motif Cys-62-Thr-Cys-64-Cys-65 decreased the activity of hydrogenase to levels no higher than 30% of those of the parental strain. H2 oxidation with the alternate electron acceptors methylene blue and benzyl viologen was decreased. H2 evolution and exchange activities were also affected. Cys-64 possibly substitutes for either Cys-62 or Cys-65, allowing for partial activity. Mutation of the codons for Cys-294 and Cys-297 to Ser codons resulted in no hydrogenase activity. The results are consistent with alterations of the ligands of FeS clusters in the HoxK subunit of hydrogenase [corrected].

Amino Acid Sequence↗

PubMed1993

In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper.

The effect of copper on the in vivo and in vitro activity of ammonia monooxygenase (AMO) from the nitrifying bacterium Nitrosomonas europaea was investigated. The addition of CuCl2 to cell extracts resulted in 5- to 15-fold stimulation of ammonia-dependent O2 consumption, ammonia-dependent nitrite production, and hydrazine-dependent ethane oxidation. AMO activity was further stimulated in vitro by the presence of stabilizing agents, including serum albumins, spermine, or MgCl2. In contrast, the addition of CuCl2 and stabilizing agents to whole-cell suspensions did not result in any stimulation of AMO activity. The use of the AMO-specific suicide substrate acetylene revealed two populations of AMO in cell extracts. The low, copper-independent (residual) AMO activity was completely inactivated by acetylene in the absence of exogenously added copper. In contrast, the copper-dependent (activable) AMO activity was protected against acetylene inactivation in the absence of copper. However, in the presence of copper both populations of AMO were inactivated by acetylene. [14C]acetylene labelling of the 27-kDa polypeptide of AMO revealed the same extent of label incorporation in both whole cells and optimally copper-stimulated cell extracts. In the absence of copper, the label incorporation in cell extracts was proportional to the level of residual AMO activity. Other metal ions tested, including Zn2+, Co2+, Ni2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Cr3+, and Ag+, were ineffective at stimulating AMO activity or facilitating the incorporation of 14C label from [14C]acetylene into the 27-kDa polypeptide. On the basis of these results, we propose that loss of AMO activity upon lysis of N. europaea results from the loss of copper from AMO, generating a catalytically inactive, yet stable and activable, form of the enzyme.

Acetylene↗

PubMed1992

Acetylene inhibition of Azotobacter vinelandii hydrogenase: acetylene binds tightly to the large subunit.

Acetylene is a slow-binding inhibitor of the Ni- and Fe-containing dimeric hydrogenase isolated from Azotobacter vinelandii. Acetylene was released from hydrogenase during the recovery from inhibition. This indicates that no transformation of acetylene to another compound occurred as a result of the interaction with hydrogenase. However, the release of C2H2 proceeds more rapidly than the recovery of activity, which indicates that release of C2H2 is not sufficient for recovery of activity. Acetylene binds tightly to native hydrogenase; hydrogenase and radioactivity coelute from a gel permeation column following inhibition with 14C2H2. Acetylene, or a derivative, remains bound to the large 65,000 MW subunit (and not to the small 35,000 MW subunit) of hydrogenase following denaturation as evidenced by SDS-PAGE and fluorography of 14C2H2-inhibited hydrogenase. This result suggests that C2H2, and by analogy H2, binds to and is activated by the large subunit of this dimeric hydrogenase. Radioactivity is lost from 14C2H2-inhibited protein during recovery. The inhibition is remarkably specific for C2H2: propyne, butyne, and ethylene are not inhibitors.

Acetylene↗

PubMed1992

Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 20.3) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Av1) were more complex than for Av2. A time-dependent inactivation of Av1 activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1992

14C2H2- and 14CO2-labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase.

Incubation of cells of the nitrifying bacterium Nitrosomonas europaea with 14C2H2 results in the covalent attachment of 14C label to a membrane-bound polypeptide of an approximate Mr of 28,000 (Hyman, M.R., and Wood, P.M. (1985) Biochem. J. 227, 719-725). A labeling procedure using 14C2H2 generated from Ba14CO3 has been used to investigate the correlation between the extent of covalent modification of this polypeptide by 14C from 14C2H2 and the level of ammonia oxidizing activity in whole cells. The time-dependent inactivation of ammonia monooxygenase by 14C2H2 resulted in a progressive and saturable incorporation of 14C into a 27-kDa polypeptide. In contrast, the specific, time-dependent and complete inactivation of ammonia monooxygenase by light resulted in concomitant decrease in the ability of cells to incorporate 14C from 14C2H2 into this polypeptide. The 14C2H2 labeling procedure was also used to investigate the recovery of ammonia monooxygenase activity after complete inactivation of pre-existing ammonia monooxygenase by either C2H2 or light. The recovery of ammonia monooxygenase activity was closely correlated with a recovery of ability of cells to incorporate 14C label from 14C2H2 into the 27-kDa polypeptide. This recovery process was energy (NH4+)-dependent and was inhibited by chloramphenicol and rifampicin, implying that de novo protein synthesis was required. Additional polypeptides labeled with 14C from 14CO2 were also identified during recovery from C2H2 or light inactivation of ammonia monooxygenase.

Acetylene↗

PubMed1992

Cometabolic degradation of chlorinated alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain.

Propylene-grown Xanthobacter cells (strain Py2) degraded several chlorinated alkenes of environmental concern, including trichloroethylene, 1-chloroethylene (vinyl chloride), cis- and trans-1,2-dichloroethylene, 1,3-dichloropropylene, and 2,3-dichloropropylene. 1,1-Dichloroethylene was not degraded efficiently, while tetrachloroethylene was not degraded. The role of alkene monooxygenase in catalyzing chlorinated alkene degradations was established by demonstrating that glucose-grown cells which lack alkene monooxygenase and propylene-grown cells in which alkene monooxygenase was selectively inactivated by propyne were unable to degrade the compounds. C2 and C3 chlorinated alkanes were not oxidized by alkene monooxygenase, but a number of these compounds were inhibitors of propylene and ethylene oxidation, suggesting that they compete for binding to the enzyme. A number of metabolites enhanced the rate of degradation of chlorinated alkenes, including propylene oxide, propionaldehyde, and glucose. Propylene stimulated chlorinated alkene oxidation slightly when present at a low concentration but became inhibitory at higher concentrations. Toxic effects associated with chlorinated alkene oxidations were determined by measuring the propylene oxidation and propylene oxide-dependent O2 uptake rates of cells previously incubated with chlorinated alkenes. Compounds which were substrates for alkene monooxygenase exhibited various levels of toxicity, with 1,1-dichloroethylene and trichloroethylene being the most potent inactivators of propylene oxidation and 1,3- and 2,3-dichloropropylene being the most potent inactivators of propylene oxide-dependent O2 uptake. No toxic effects were seen when cells were incubated with chlorinated alkenes anaerobically, indicating that the product(s) of chlorinated alkene oxidation mediates toxicity.

Alkenes↗

PubMed1992

The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase.

The roles of the product of the hoxZ gene immediately downstream of the hydrogenase gene (hoxKG) in Azotobacter vinelandii were investigated by constructing and characterizing a mutant with the center of the hoxZ gene deleted. The strain lacking the functional hoxZ gene product exhibited a low rate of H2 oxidation with O2 as the electron acceptor relative to that of the wild-type strain. Nevertheless, when the enzyme was exogenously activated and methylene blue was used as the electron acceptor from hydrogenase, rates of H2 oxidation comparable to those in the wild-type strain were observed. These results suggest that the gene product of hoxZ plays a role in activating and maintaining hydrogenase in a reduced active state. The product of hoxZ could also be the linkage necessary for transfer of electrons from H2 to the electron transport chain.

Azotobacter vinelandii↗

PubMed1991

Kinetic analysis of the interaction of nitric oxide with the membrane-associated, nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii.

The effects of nitric oxide (NO) on the membrane-associated form of the nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii have been investigated. In the presence of H2 and an electron acceptor (turnover conditions), NO acts as a noncompetitive inhibitor vs. methylene blue (Ki = 12 microM). There is no element of competition between NO and H2, implying that the site of NO action is not the H2-activating site of the hydrogenase. When the membrane-associated hydrogenase is incubated under non-turnover conditions, the enzyme is irreversibly inactivated by NO in a time-dependent process. The inactivation is a non-saturable, pseudo-first-order process which is consistent with a direct chemical reaction between NO and the hydrogenase. Kinetic evidence is presented which is compatible with an interaction between NO and a redox-active component other than the H2-activating site on the enzyme. The complex inhibition pattern of NO has been interpreted in terms of two distinct interactions of NO with iron-sulfur centers of the hydrogenase.

Azotobacter↗

PubMed1991

Aerobically purified hydrogenase from Azotobacter vinelandii: activity, activation, and spectral properties.

The hydrogenase from Azotobacter vinelandii is typically purified under anaerobic conditions. In this work, the hydrogenase was purified aerobically. The yields were low (about 2%) relative to those of the anaerobic purification (about 20%). The rate of enzyme activity depended upon the history of the enzyme. The enzyme preparations were active as isolated in H2 oxidation, and isotope exchange. The activity increased during the assay to a new maximal level (turnover activation). Treatment with reductants (e.g., H2, dithionite, dithiothreitol, indigo carmine) resulted in greater activation (reductant activation). Activation of the hydrogenase was accompanied by decrease in visible light absorption (300-600 nm) with maximal decreases at 450 and 345 nm which indicated the reduction of iron-sulfur clusters. The aerobically purified hydrogenase was susceptible to irreversible inactivation by cyanide. Pretreatment with acetylene did not influence activation of the hydrogenase. Once activated, the aerobically purified hydrogenase was indistinguishable from the anaerobically purified hydrogenase with respect to the catalytic properties tested.

Acetylene↗

PubMed1991

Factors Limiting Aliphatic Chlorocarbon Degradation by Nitrosomonas europaea: Cometabolic Inactivation of Ammonia Monooxygenase and Substrate Specificity.

The soil nitrifying bacterium Nitrosomonas europaea is capable of degrading trichloroethylene (TCE) and other halogenated hydrocarbons. TCE cometabolism by N. europaea resulted in an irreversible loss of TCE biodegradative capacity, ammonia-oxidizing activity, and ammonia-dependent O(2) uptake by the cells. Inactivation was not observed in the presence of allylthiourea, a specific inhibitor of the enzyme ammonia monooxygenase, or under anaerobic conditions, indicating that the TCE-mediated inactivation required ammonia monooxygenase activity. When N. europaea cells were incubated with [C]TCE under conditions which allowed turnover of ammonia monooxygenase, a number of cellular proteins were covalently labeled with C. Treatment of cells with allylthiourea or acetylene prior to incubation with [C]TCE prevented incorporation of C into proteins. The ammonia-oxidizing activity of cells inactivated in the presence of TCE could be recovered through a process requiring de novo protein synthesis. In addition to TCE, a series of chlorinated methanes, ethanes, and other ethylenes were screened as substrates for ammonia monooxygenase and for their ability to inactivate the ammonia-oxidizing system of N. europaea. The chlorocarbons could be divided into three classes depending on their biodegradability and inactivating potential: (i) compounds which were not biodegradable by N. europaea and which had no toxic effect on the cells; (ii) compounds which were cooxidized by N. europaea and had little or no toxic effect on the cells; and (iii) compounds which were cooxidized and produced a turnover-dependent inactivation of ammonia oxidation by N. europaea.

Journal Article↗

PubMed1990

The small-scale production of [U-14C]acetylene from Ba14CO3: application to labeling of ammonia monooxygenase in autotrophic nitrifying bacteria.

A small-scale method has been adapted from an established procedure for the generation of [U-14C]acetylene from inexpensive and commonly available precursors. The method involves the fusing of Ba14CO3 with excess barium metal to produce Ba14C2. The BaC2 is reacted with water to generate acetylene which is then selectively dissolved into dimethyl sulfoxide (DMSO). The results presented demonstrate the effect of Ba:BaCO3 ratio on the concentrations of various gases released during the hydrolysis reaction and quantify the selectivity of the DMSO-trapping process for each gas. [U-14C]Acetylene generated by this method has been used to inactivate ammonia monooxygenase in three species of autotrophic nitrifying bacteria: Nitrosomonas europaea, Nitrosococcus oceanus, and Nitrosolobus multiformis. Our results demonstrate that acetylene inactivation of this enzyme in all three species results in the covalent incorporation of radioactive label into a polypeptide of apparent Mr of 25,000-27,000, as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography.

Acetylene↗

PubMed1990

The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum.

The structural genes (hupSL) of the membrane-bound NiFe-containing H2-uptake hydrogenase (Hup) of Azotobacter chroococcum were identified by oligonucleotide screening and sequenced. The small subunit gene (hupS) encodes a signal sequence of 34 amino acids followed by a 310-amino-acid, 34156D protein containing 12 cysteine residues. The large subunit gene (hupL) overlaps hupS by one base and codes for a predicted 601-amino-acid, 66433D protein. There are two regions of strong homology with other Ni hydrogenases: a Cys-Thr-Cys-Cys-Ser motif near the N-terminus of HupS and an Asp-Pro-Cys-Leu-Ala-Cys motif near the carboxy-terminus of HupL. Strong overall homology exists between Azotobacter, Bradyrhizobium japonicum and Rhodobacter capsulatus Hup proteins but less exists between the Azotobacter proteins and hydrogenases from Desulfovibrio strains. Mutagenesis of either hupS or hupL genes of A. chroococcum yielded Hup- phenotypes but some of these mutants retained a partial H2-evolving activity. Hybridization experiments at different stages of gene segregation confirmed the multicopy nature of the Azotobacter genome.

Amino Acid Sequence↗

PubMed1990

Inhibition of ammonia monooxygenase in Nitrosomonas europaea by carbon disulfide.

Carbon disulfide has long been recognized as a potent inhibitor of nitrification, and it is the likely active component in several nitrification inhibitors suitable for field use. The effects of this compound on Nitrosomonas europaea have been investigated, and the site of action has been determined. Low concentrations of CS2 (less than 400 microM) produced a time-dependent inhibition of ammonia-dependent O2 uptake but did not inhibit hydrazine-oxidizing activity. CS2 also produced distinct changes in difference spectra of whole cells. These results suggest that ammonia monooxygenase (AMO) is the site of action of CS2. Unlike the case for thiourea and acetylene, saturating concentrations of CS2 did not fully inhibit AMO, and the inhibition resulted in a low but significant rate of ammonia-dependent O2 uptake. The effects of CS2 were not competitive with respect to ammonia concentration, and the inhibition by CS2 did not require the turnover of AMO to take effect. The ability of CS2-treated cells to incorporate [14C]acetylene into the 28-kilodalton polypeptide of AMO was used to demonstrate that the effects of CS2 are compatible with a mode of action which involves a reduction of the rate of turnover of AMO without effects on the catalytic mechanism. It is proposed that CS2 may act on AMO by reversibly reacting with a suitable nucleophilic amino acid in close proximity to the active site copper.

Acetylene↗

PubMed1990

Oxidation of monohalogenated ethanes and n-chlorinated alkanes by whole cells of Nitrosomonas europaea.

We have investigated the substrate specificity of ammonia monooxygenase in whole cells of the nitrifying bacterium Nitrosomonas europaea for a number of aliphatic halogenated hydrocarbons. To determine the effect of the halogen substituent and carbon chain length on substrate reactivity, we measured the rates of oxidation of the monohalogenated ethanes (fluoroethane, chloroethane, bromoethane, and iodoethane) and n-chlorinated C1 to C4 alkanes by whole cells of N. europaea. For monohalogenated ethanes, acetaldehyde was the major organic product and little or none of any of the alternate predicted products (2-halogenated alcohols) were detected. The maximum rate of haloethane oxidation increased with decreasing halogen molecular weight from iodoethane to chloroethane (19 to 221 nmol/min per mg of protein). In addition, the amount of substrate required for the highest rate of haloethane oxidation increased with decreasing halogen molecular weight. For the n-chlorinated alkanes, the rate of dechlorination, as measured by the appearance of the corresponding aldehyde product, was greatest for chloroethane and decreased dramatically for chloropropane and chlorobutane (118, 4, and 8 nmol of aldehyde formed per min per mg of protein, respectively). The concentration profiles for halocarbon oxidation by ammonia monooxygenase showed apparent substrate inhibition when ammonia was used as the reductant source. When hydrazine was used as the electron donor, no substrate inhibition was observed, suggesting that the inhibition resulted from reductant limitation.

Alkanes↗