Differential inhibition in vivo of ammonia monooxygenase, soluble methane monooxygenase and membrane-associated methane monoxygenase by phenylacetylene

Phenylacetylene was investigated as a differential inhibitor of ammonia monooxygenase (AMO), soluble methane monooxygenase (sMMO) and membrane-associated or particulate methane monooxygenase (pMMO) in vivo. At phenylacetylene concentrations > 1 microM, whole-cell AMO activity in Nitrosomonas europaea was completely inhibited. Phenylacetylene concentrations above 100 microM inhibited more than 90% of sMMO activity in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b. In contrast, activity of pMMO in M. trichosporium OB3b, M. capsulatus Bath, Methylomicrobium album BG8, Methylobacter marinus A45 and Methylomonas strain MN was still measurable at phenylacetylene concentrations up to 1,000 microM. AMO of Nitrosococcus oceanus has more sequence similarity to pMMO than to AMO of N. europaea. Correspondingly, AMO in N. oceanus was also measurable in the presence of 1,000 microM phenylacetylene. Measurement of oxygen uptake indicated that phenylacetylene acted as a specific and mechanistic-based inhibitor of whole-cell sMMO activity; inactivation of sMMO was irreversible, time dependent, first order and required catalytic turnover. Corresponding measurement of oxygen uptake in whole cells of methanotrophs expressing pMMO showed that pMMO activity was inhibited by phenylacetylene, but only if methane was already being oxidized, and then only at much higher concentrations of phenylacetylene and at lower rates compared with sMMO. As phenylacetylene has a high solubility and low volatility, it may prove to be useful for monitoring methanotrophic and nitrifying activity as well as identifying the form of MMO predominantly expressed in situ.     

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.     

Major changes in copper coordination accompany reduction of peptidylglycine monooxygenase: implications for electron transfer and the catalytic mechanism

H and Cu_M). The Cu_H center changes from 4- or 5-coordinate tetragonal to a 2-coordinate configuration, with one of the three histidine ligands becoming undetectable by EXAFS (suggesting that it has moved away from the Cu_H by at least 0.3 Å). The Cu_M center changes from 4- or 5-coordinate tetragonal to a trigonal or tetrahedral configuration, with an estimated 0.3–0.5 Å movement of the M314 S ligand. Reduction also leads to loss of coordinated water from both of the coppers. Substrate binding has little or no effect on the local environment of the Cu centers in either oxidation state. These findings bring into question whether direct electron transfer between Cu_H and Cu_M via a tunneling mechanism can be fast enough to support the observed catalytic rate, and suggest that some other mechanism for electron transfer, such as superoxide channeling, should be considered. Received: 17 November 1999 / Accepted: 25 February 2000     

Structural conservation of the B subunit in the ammonia monooxygenase/particulate methane monooxygenase superfamily

The ammonia monooxygenase (AMO)/particulate methane monooxygenase (pMMO) superfamily is a diverse group of membrane‐bound enzymes of which only pMMO has been characterized on the molecular level. The pMMO active site is believed to reside in the soluble N‐terminal region of the pmoB subunit. To understand the degree of structural conservation within this superfamily, the crystal structure of the corresponding domain of an archaeal amoB subunit from Nitrosocaldus yellowstonii has been determined to 1.8 Å resolution. The structure reveals a remarkable conservation of overall fold and copper binding site location as well as several notable differences that may have implications for function and stability. Proteins 2014; 82:2263–2267. © 2014 Wiley Periodicals, Inc.     

Substrate specificity of soluble methane monooxygenase. Mechanistic implications

Following the example set by studies of the mechanistic aspects of the substrate specificity of various cytochrome P-450 enzymes, we have undertaken a parallel investigation of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme with a broad substrate specificity. Using substrates previously tested with cytochrome P-450 enzymes and using purified enzyme preparations, this work indicates that soluble methane monooxygenase has a similar oxidative reaction mechanism to cytochrome P-450 enzymes. The evidence suggests that soluble methane monooxygenase oxidizes substrates via a nonconcerted reaction mechanism (hydrogen abstraction preceding hydroxylation) with radical or carbocation intermediates. Aromatic hydroxylation proceeds by epoxidation followed by an NIH shift.     

Soluble methane monooxygenase: activation of dioxygen and methane

The mechanisms by which soluble methane monooxygenase uses dioxygen to convert methane selectively to methanol have come into sharp focus. Diverse techniques have clarified subtle details about each step in the reaction, from binding and activating dioxygen, to hydroxylation of alkanes and other substrates, to the electron transfer events required to complete the catalytic cycle.     

Inhibition by ammonia of methane utilization in Methylococcus capsulatus (Bath)

Summary The kinetics of methane uptake by Methylococcus capsulatus (Bath) and its inhibition by ammonia were studied by stopped-flow membrane-inlet mass spectrometry. Measurements were done on suspensions of cells grown in high- and low-copper media. With both types of cells the kinetics of methane uptake are hyperbolic when oxygen is in excess. The apparent K _m and K _max for methane uptake are both higher in low-copper cells than in high-copper cells. Ammonia is a simple competitive inhibitor of methane uptake in high-copper cells when the oxygen concentration is above a few M. The findings agree with the assumption that ammonia is a week alternative substrate for particulate methane monooxygenase. In low-copper cells the effect of ammonia is complicated and cannot be explained in terms of current assumptions on the mechanism of soluble methane monooxygenase. Our data indicate that ammonia inhibition is likely to be a more serious problem in connection with cultivation in low-copper medium than in high-copper medium. Offprint requests to: H. N. Carlsen     

Inhibition of photosynthetic electron transfer by amphotericin B

The polyene antibiotic amphotericin B inhibits photosynthetic electron transfer by Class II maize mesophyll chloroplasts, from water to FeCN, DCIP and diquat but not to plastocyanin. Photosystem 1 activity is also inhibited by amphotericin B, but ferredoxin-NADP reductase activity is not affected. The activity of all the photosynthetic electron transfer systems inhibited by amphotericin B can be restored by the addition of carrier amounts of plastocyanin. The results suggest that amphotericin B inhibits photosynthetic electron transfer by acting only at the plastocyanin site in the chain, and that the primary site of reduction of FeCN and DCIP from water by Class II chloroplasts lies on the reducing side of photosystem 1.     

Evidence that a type-2 NADH:quinone oxidoreductase mediates electron transfer to particulate methane monooxygenase in methylococcus capsulatus.

NADH readily provides reducing equivalents to membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) in isolated membrane fractions, but detergent solubilization disrupts this electron-transfer process. Addition of exogenous quinones (especially decyl-plastoquinone and duroquinone) restores the NADH-dependent pMMO activity. Results of inhibitor and substrate dependence of this activity indicate the presence of only a type-2 NADH:quinone oxidoreductase (NDH-2). A 100-fold purification of the NDH-2 was achieved using lauryl-maltoside solubilization followed by ion exchange, hydrophobic-interaction, and gel-filtration chromatography. The purified NDH-2 has a subunit molecular weight of 36 kDa and exists as a monomer in solution. UV-visible and fluorescence spectroscopy identified flavin adenine dinucleotide (FAD) as a cofactor present in stoichiometric amounts. NADH served as the source of electrons, whereas NADPH could not. The purified NDH-2 enzyme reduced coenzyme Q(0), duroquinone, and menaquinone at high rates, whereas the decyl analogs of ubiquinone and plastoquinone were reduced at approximately 100-fold lower rates. Rotenone and flavone did not inhibit the NDH-2, whereas amytal caused partial inhibition but only at high concentrations.     

Tri- and tetramethylhydroquinone as electron donors for ammonia monooxygenase in whole cells of Nitrosomonas europaea

Abstract Ten redox reagents have been tested as electron donors to ammonia monooxygenase in whole cells of Nitrosomonas europaea . Positive results were obtained with tri- and tetramethylhydroquinone. An earlier study showed that phenol was converted into hydroquinone by the monooxygenase. Cells were therefore incubated with trimethylphenol, to see if its hydroxylation to trimethylhydroquinone would lead to a self-sufficient conversion of trimethylphenol into trimethylquinone. No trimethylquinone could be detected. The maximal rates of propene epoxidation obtained with tri-and tetramethylhydroquinone were 1.8 and 4.6 μmol · h⁻¹· mg protein⁻¹, respectively.     

Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M

The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out.     

Haloalkene oxidation by the soluble methane monooxygenase from Methylosinus trichosporium OB3b: mechanistic and environmental implications

The soluble, three-protein component methane monooxygenase purified from Methylosinus trichosporium OB3b is capable of oxidizing chlorinated, fluorinated, and brominated alkenes, including the widely distributed ground-water contaminant trichloroethylene (TCE). The oxidation rates for the chloroalkenes were observed to be comparable to that for methane, the natural substrate, and up to 7000-fold higher than those reported for other well-defined biological systems. The competitive inhibitor tetrachloroethylene was found to be the only chlorinated ethylene not turned over. However, this appears to be due to steric effects rather than electronic effects or the lack of an abstractable proton because chlorotrifluoroethylene was efficiently oxidized. As evidenced by the formation of diagnostic adducts with 4-(p-nitrobenzyl)pyridine, the halogenated alkenes were oxidized predominantly by epoxidation. Stable acidic products resulting from subsequent hydrolysis were identified as the major products. However, additional aldehydic products resulting from intramolecular halide or hydride migration were observed in 3-10% yield during the oxidation of TCE, vinylidene chloride, trifluorethylene, and tribromoethylene. Product analysis of the hydrolysis reaction of authentic TCE epoxide showed little or no 2,2,2-trichloroacetaldehyde (chloral) formation, indicating that atomic migration occurred prior to product dissociation from the enzyme. The occurrence of atomic migration products shows that an intermediate in the substrate to product conversion carries significant cationic character. Such a species could be generated through interaction with a highly electron-deficient activated oxygen in the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Method for assaying methane monooxygenase activity by measuring methane uptake in the reaction vessel

The reaction vessel has been designed to measure methane monooxygenase activity. An elastic membrane has been built into one of the walls of the vessel to take liquid samples, avoiding formation of the gaseous phase in the reaction volume. The methane content in the samples is measured in a gas-liquid chromatograph with a flame ionization detector in two ways: 1. by direct measurement of methane in the liquid sample, and 2. by measurement of methane in the gaseous phase after methane diffusion from the liquid sample into the gaseous space of another vessel. The method is simple, sensitive (with a lower limit of 0.1 nMole CH4), and well reproducible. This method permits measurement of the oxidation kinetics of methane and other gaseous hydrocarbons both by intact cells and cell-free preparations of methane oxidizing bacteria.     

Oxidation of deuterated compounds by high specific activity methane monooxygenase from Methylosinus trichosporium. Mechanistic implications

Hydrocarbon oxidations catalyzed by methane monooxygenase purified to high specific activity from the type II methanotroph Methylosinus trichosporium OB3b were compared to the same reactions catalyzed by methane monooxygenase from the type I methanotroph Methylococcus capsulatus Bath and liver microsomal cytochrome P-450. The two methane monooxygenases produced nearly identical product distributions, in accord with physical studies of the enzymes which have shown them to be very similar. The products obtained from the oxidation of a series of deuterated substrates by the M. trichosporium methane monooxygenase were very similar to those reported for the same reaction catalyzed by liver microsomal cytochrome P-450, suggesting that the enzymes use similar mechanisms. However, differences in the product distributions and other aspects of the reactions indicated the mechanisms are not identical. Methane monooxygenase epoxidized propene in D2O and d6-propene in H2O without exchange of substrate protons or deuterons with solvent, in contrast to cytochrome P-450 (Groves, J. T., Avaria-Neisser, G. E., Fish, K. M., Imachi, M., and Kuczkowski, R. L. (1986) J. Am. Chem. Soc. 108, 3837-3838), suggesting that the mechanism of epoxidation of olefins by methane monooxygenase differs at least in part from that of cytochrome P-450. Hydroxylation of alkanes by methane monooxygenase revealed close similarities to hydroxylations by cytochrome P-450. Allylic hydroxylation of 3,3,6,6-d4-cyclohexene occurred with approximately 20% allylic rearrangement in the case of methane monooxygenase, whereas 33% was reported for this reaction catalyzed by cytochrome P-450 (Groves, J. T., and Subramanian, D. V. (1984) J. Am. Chem. Soc. 106, 2177-2181). Similarly, hydroxylation of exo,exo,exo,exo-2,3,5,6-d4-norbornane by methane monooxygenase occurred with epimerization, but to a lesser extent than reported for cytochrome P-450 (Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J. (1978) Biochem. Biophys. Res. Commun. 81, 154-160). A large intramolecular isotope effect, kH,exo/kD,exo greater than or equal to 5.5, was calculated for this reaction. However, the intermolecular kinetic isotope effect on Vm for methane oxidation was small, suggesting that steps other than C-H bond breakage were rate limiting in the overall enzymatic reaction. Similar isotope effects have been observed for cytochrome P-450. These observations indicate a stepwise mechanism of hydroxylation for methane monooxygenase analogous to that proposed for cytochrome P-450.(ABSTRACT TRUNCATED AT 400 WORDS)     

Positively charged amino acids are essential for electron transfer and protein-protein interactions in the soluble methane monooxygenase complex from Methylococcus capsulatus (Bath)

The soluble methane monooxygenase (sMMO) complex from Methylococcus capsulatus (Bath) catalyses oxygen- and NAD(P)H-dependent oxygenation of methane, propene, and other substrates. Whole-complex sMMO oxygenase activity requires all three sMMO components: the hydroxylase, the reductase, and protein B. Also, in the presence of hydrogen peroxide, the hydroxylase alone catalyzes substrate oxygenation via the peroxide shunt reaction. We investigated the effect of amine cross-linking on hydroxylase activity to probe the role of a gross conformational change that occurs in the hydroxylase upon binding of the other protein components. The cross-linker inhibited hydroxylase activity in the whole complex, but this effect was due to covalent modification of primary amine groups rather than cross-linking. Covalent modification of arginine side-chains on the hydroxylase had a similar effect, but, most remarkably, neither form of modification affected the activity of the hydroxylase via the peroxide shunt reaction. It was shown that covalent modification of positively charged groups on the hydroxylase, which occurred at multiple sites, interfered with its physical and functional interactions with protein B and with the passage of electrons from the reductase. These results indicate that protein B and the reductase of the sMMO complex interact via positively charged groups on the surface of the hydroxylase to induce a conformational change that is necessary for delivery of electrons into the active site of the hydroxylase. Modification of positively charged groups on protein B had no effect on its function, consistent with the hypothesis that positively charged groups on the hydroxylase interact with negative charges on protein B. Thus, we have discovered a means of specifically inactivating the interactions between the sMMO complex while preserving the catalytic activity of the hydroxylase active site which provides a new method of studying intercomponent interactions within sMMO.     

Inhibition of membrane-bound succinate dehydrogenase by disulfiram

The effect of disulfiram on succinate oxidase and succinate dehydrogenase activities of beef heart submitochondrial particles was studied. Results show that disulfiram inhibits both functions. Succinate and malonate suppress the inhibitory action of disulfiram when succinate dehydrogenase is stabilized in an active conformation. Disulfiram is not able to inhibit the enzyme when succinate dehydrogenase is inactivated by oxaloacetate. The inhibitory effect of disulfiram is reverted by the addition of dithiothreitol. From these results, it is proposed that disulfiram inhibits the utilization of succinate by a direct modification of an -SH group located in the catalytically active site of succinate dehydrogenase.     

Inhibition of membrane-bound succinate dehydrogenase by fluorescamine

Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.     

Molecular biology and regulation of methane monooxygenase

Methanotrophs are ubiquitous in the environment and play an important role in mitigating global warming due to methane. They are also potentially interesting for industrial applications such as production of bulk chemicals or bioremediation. The first step in the oxidation of methane is the conversion to methanol by methane monooxygenase, the key enzyme, which exists in two forms: the cytoplasmic, soluble methane monooxygenase (sMMO) and the membrane-bound, particulate methane monooxygenase (pMMO). This paper reviews the biochemistry and molecular biology of both forms of MMO. In the past few years there have been many exciting new findings. sMMO components have been expressed in heterologous and homologous hosts. The pMMO has been purified and biochemically studied in some detail and the genes encoding the pMMO have been sequenced. Copper ions have been shown to play a key role in regulating the expression of both MMO enzyme complexes. We also present a model for copper regulation based on results from Northern analysis, primer-extensions and new sequence data, and raise a number of unanswered questions for future studies.