Substrate radical intermediates in soluble methane monooxygenase

EPR spin-trapping experiments were carried out using the three-component soluble methane monooxygenase (MMO). Spin-traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), alpha-4-pyridyl-1-oxide N-tert-butylnitrone (POBN), and nitrosobenzene (NOB) were used to investigate the possible formation of substrate radical intermediates during catalysis. In contrast to a previous report, the NADH-coupled oxidations of various substrates did not produce any trapped radical species when DMPO or POBN was present. However, radicals were detected by these traps when only the MMO reductase component and NADH were present. DMPO and POBN were found to be weak inhibitors of the MMO reaction. In contrast, NOB is a strong inhibitor for the MMO-catalyzed nitrobenzene oxidation reaction. When NOB was used as a spin-trap in the complete MMO system with or without substrate, EPR signals from an NOB radical were detected. We propose that a molecule of NOB acts simultaneously as a substrate and a spin-trap for MMO, yielding the long-lived radical and supporting a stepwise mechanism for MMO.     

Substrate binding and C-H bond activation in the soluble methane monooxygenase hydroxylase

 The selective oxidation of CH₄ to CH₃OH is a conceptually simple, yet functionally difficult, chemical transformation. In nature, this reaction is performed by methane monooxygenases, the soluble class of which employ carboxylate-bridged dinuclear iron centers to activate dioxygen. The process by which small molecules access the active site of the sMMO hydroxylase, the structures of intermediates in the catalytic reaction cycle, and mechanistic details about the attack on the C–H bond are subjects of intense investigation. In this commentary, we present our current views on exogenous ligand binding and dioxygen activation at the active site and the mechanism of alkane hydroxylation. Received: 20 October 1997 / Accepted: 26 February 1998     

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)     

Desaturation reactions catalyzed by soluble methane monooxygenase

Soluble methane monooxygenase (MMO) is shown to be capable of catalyzing desaturation reactions in addition to the usual hydroxylation and epoxidation reactions. Dehydrogenated products are generated from MMO-catalyzed oxidation of certain substrates including ethylbenzene and cyclohexadienes. In the reaction of ethylbenzene, desaturation of ethyl C-H occurred along with the conventional hydroxvlations of ethyl and phenyl C-Hs. As a result, styrene is formed together with ethylphenols and phenylethanols. Similarly, when 1,3- and 1,4-cyclohexadienes were used as substrates, benzene was detected as a product in addition to the corresponding alcohols and epoxides. In all cases, reaction conditions were found to significantly affect the distribution among the different products. This new activity of MMO is postulated to be associated with the chemical properties of the substrates rather than fundamental changes in the nature of the oxygen and C-H activation chemistries. The formation of the desaturated products is rationalized by formation of a substrate cationic intermediate, possibly via a radical precursor. The cationic species is then proposed to partition between recombination (alcohol formation) and elimination (alkene production) pathways. This novel function of MMO indicates close mechanistic kinship between the hydroxylation and desaturation reactions catalyzed by the nonheme diiron clusters.     

Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase

The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.     

Oxygen kinetic isotope effects in soluble methane monooxygenase

Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridged diiron site that activates dioxygen in the catalytic oxidation of hydrocarbon substrates. Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH(3)CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which produced glycolonitrile (HOCH(2)CN) and uncoupled oxidase activity that led to water formation. The oxygen KIE was measured independently for both the oxygenase and oxidase reactions, and values of 1.0152 +/- 0.0007 and 1.0167 +/- 0.0010 were obtained, respectively. The isotope effects and separate dioxygen binding studies do not support irreversible formation of an enzyme-dioxygen Michaelis complex. Additional mechanistic implications are discussed in the context of previous data obtained from single turnover and steady-state kinetic studies.     

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.     

Screening of obligate methanotrophs for soluble methane monooxygenase genes

A 5.8 kb fragment of chromosomal DNA from Methylococcus capsulatus (Bath) containing genes encoding the soluble methane monooxygenase enzyme complex was used as a probe for the detection of soluble monooxygenase genes in a number of representative strains of obligate methanotrophs. Only type II methanotrophs of the genus Methylosinus were found to contain homologues to the Methylococcus gene probe. This probe was also used successfully to detect soluble methane monooxygenase genes in a variety of methanotrophs by colony hybridizations.     

Applications of a colorimetric plate assay for soluble methane monooxygenase activity.

A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in the development of a colored complex between 1-naphthol, which is formed when sMMO reacts with naphthalene, and o-dianisidine (tetrazotized). Methanotrophic colonies expressing sMMO turned deep purple when exposed successively to naphthalene and o-dianisidine. The method was evaluated within the contexts of two potential applications. The first was for the enumeration of Methylosinus trichosporium OB3b in a methane-amended, unsaturated soil column dedicated to vinyl chloride treatment. The second application was for the isolation and enumeration of sMMO-bearing methanotrophs from sanitary landfill soils. The technique was effective in both applications.     

Applications of a colorimetric plate assay for soluble methane monooxygenase activity.

A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in the development of a colored complex between 1-naphthol, which is formed when sMMO reacts with naphthalene, and o-dianisidine (tetrazotized). Methanotrophic colonies expressing sMMO turned deep purple when exposed successively to naphthalene and o-dianisidine. The method was evaluated within the contexts of two potential applications. The first was for the enumeration of Methylosinus trichosporium OB3b in a methane-amended, unsaturated soil column dedicated to vinyl chloride treatment. The second application was for the isolation and enumeration of sMMO-bearing methanotrophs from sanitary landfill soils. The technique was effective in both applications.     

pH Dependence of peptidylglycine monooxygenase. Mechanistic implications of Cu-methionine binding dynamics

The pH dependence of the PHM-catalyzed monooxygenation of dansyl-YVG was studied in two different buffer systems in the pH range of 4-10. The pH-activity profile measured in a sulfonic acid buffer exhibited a maximum at pH 5.8 and became inactive at pH >9. The data could be fit to a model that assumed a protonated unreactive species A, a major reactive species B, and a less reactive species C. B formed in a deprotonation step with pK(a) of 4.6, while C formed and decayed with pK(a)s of 6.8 and 8.2, respectively. The pH dependence was found to be dominated by k(cat), with K(m)(dansyl-YVG) remaining pH-independent over the pH range of 5-8. Acetate-containing buffers shifted the pH maximum to 7.0, and the activity-pH profile could be simulated by formation and decay of a single active species with pK(a)s of 5.8 and 8.3, respectively. The pH-dependent changes in activity could be correlated with a change in the Debye-Waller factor for the Cu-S(met) (M314) component of the X-ray absorption spectrum which underwent a transition from a tightly bound inactive "met-on" form to a conformationally mobile active "met-off" form with a pK(a) which tracked the formation of the active species in both sulfonic acid and acetate-containing buffer systems. The data suggested that the conformational mobility of the bound substrate relative to the copper-superoxo active species is critical to catalysis and further suggested the presence of an accessible vibrational mode coupling Cu-S motion to the H tunneling probability along the Cu-O...H...C coordinate.     

Substrate specificity of soluble mitochondrial ATPase]

The parameters of the hydrolysis of ATP and several analogs by soluble mitochondrial ATPase were determined. Vmax of the reaction decreases within the range: 2'-desoxy-ATP greater than ATP greater than etheno-ATP greater than GTP greater than 3'-O-methylATP greater than UTP. ATP, 2'-desoxypATP, 3'O-methyl-ATP, GTP, and etheno-ATP are hydrolysed by soluble mitochondrial ATPase with close Km(app) values. CTP is not hydrolysed by the enzyme and does not inhibit the ATPase reaction at a concentration of 10(-2) M. Nucleoside triphosphate derivatives with an "open" ribose cycle 9-[1',5'-dihydroxy-4-(S)-hydroxymethyl-3'-oxapent-2' (R)-yl]adenyl-5'-triphosphate, and 1-[1',5'-dihydroxy-4'-(S)-hydroxymethyl-3'-oxapent-2'(R)-yl[cytosine-5'-triphosphate are effective inhibitors of ATPase (Ki approximately 5.10(-5)M). Mitochondrial ATPase binds the ATP analogs that have hydrocarbon radicals-(CH2)2-, -(CH2)3-, and (CH2)4- instead of the ribose residues: 9-(2'hydroxyethyl)adenyl-2'-triphosphate, 9-(3'-hydroxypropyl)-adenine-3'-triphosphate, and 9-(4'-hydroxybutyl)adenine-4'-triphosphyl)adenine-4'-triphosphate were not hydrolysed by the enzyme, although they inbibit the ATPase reaction (Ki 2.10(-4)M). 9-(2'-hydroxyethyl)adenine-2'-triphosphate is hydrolysed by ATPase eight times more slowly than ATP. It is suggested that the hydrolysis of the substrates of mitochondrial ATPase is- preceded by the binding of the substrates in a tense conformation in the active site of the enzyme.     

A rapid fluorescence-based assay for detecting soluble methane monooxygenase

A fluorescence-based assay was developed to estimate soluble methane monooxygenase (sMMO) activity in solution. Whole cells of Methylosinus trichosporium OB3b expressing sMMO were used to oxidize various compounds to screen for fluorescent products. Of the 12 compounds tested, only coumarin yielded a fluorescent product. The UV absorbance spectrum of the product matches that of 7-hydroxycoumarin, and this identification was confirmed by 13C-NMR spectroscopy. The dependence of the fluorescent reaction on sMMO activity was investigated by pre-incubation with acetylene, a known inhibitor of sMMO activity. Apparent kinetic parameters for whole cells were determined to be Km(app)=262 microM and Vmax(app)=821 nmol 7-hydroxycoumarin min(-1) mg protein(-1). The rate of coumarin oxidation by sMMO correlates well with those of trichloroethylene degradation and naphthalene oxidation. Advantages of the fluorescence-based coumarin oxidation assay over the naphthalene oxidation assay include a more stable product, direct detection of the product without additional reagents, and greater speed and convenience.     

Applications of a colorimetric plate assay for soluble methane monooxygenase activity

[This corrects the article on p. 2235 in vol. 58.].     

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)     

Comparative characteristics of soluble and membrane brain aminopeptidases. II. Substrate specificity

Comparative studies on substrate specificity of the soluble and membrane-bound aminopeptidases from bovine brain were carried out. A series of p-nitroanilides and beta-naphthylamides of amino acids, di- and tripeptides with the aminoterminal phenylalanine residue, as well as a biologically active pentapeptide--[Leu5]enkephalin--were used as substrates. The soluble and membrane-bound aminopeptidases manifested identical specificity towards the employed substrates. The aminopeptidases were equally effective towards the p-nitroanilides of amino acids and peptides, whereas beta-naphthylamides were more susceptible to hydrolysis by both aminopeptidases than p-nitroanilides and peptides. Taking into account physico-chemical characteristics of these enzymes, it was concluded that the soluble and membrane-bound aminopeptidases are quite similar or perhaps identical. Their role in the regulation of nervous system functioning was discussed. A comparison of specificities for brain aminopeptidases and leucine aminopeptidase from bovine lens led to the conclusion that they belong to different groups. This feature allows planning the synthesis of selective inhibitors.     

Two-step concerted mechanism for methane hydroxylation on the diiron active site of soluble methane monooxygenase

A new concerted mechanism is proposed for the conversion of methane to methanol on intermediate Q of soluble methane monooxygenase (sMMO), the active site of which is considered to involve an Fe2(mu-O)2 diamond core. A hybrid density functional theory (DFT) method is used for our mechanistic study on the important reactivity of the bare FeO+ complex and a diiron model of intermediate Q. The reaction pathway for the methane hydroxylation on the diiron complex is essentially identical to that for the gas-phase reaction by the bare FeO+ complex. Methane is highly activated on the dinuclear iron model through the formation of a methane complex, in which a coordinatively unsaturated iron plays a central role in the bonding interaction between the diiron model and substrate methane. A H atom abstraction via a four-centered transition state and a recombination of the OH and CH3 groups via a three-centered transition state successively occur on the dinuclear iron-oxo species, leading to the formation of a methanol complex that corresponds to intermediate T. These electronic processes take place in a concerted manner. Our mechanism for methane hydroxylation by sMMO is different from the radical mechanism that has been widely accepted for enzymatic hydrocarbon hydroxylation, especially by cytochrome P450.     

The soluble methane monooxygenase gene cluster of the trichloroethylene-degrading methanotroph Methylocystis sp. strain M.

In methanotrophic bacteria, methane is oxidized to methanol by the enzyme methane monooxygenase (MMO). The soluble MMO enzyme complex from Methylocystis sp. strain M also oxidizes a wide range of aliphatic and aromatic compounds, including trichloroethylene. In this study, heterologous DNA probes from the type II methanotroph Methylosinus trichosporium OB3b were used to isolate souble MMO (sMMO) genes from the type II methanotroph Methylocystis sp. strain M. sMMO genes from strain M are clustered on the chromosome and show a high degree of identity with the corresponding genes from Methylosinus trichosporium OB3b. Sequencing and phylogenetic analysis of the 16S rRNA gene from Methylocystis sp. strain M have confirmed that it is most closely related to the type II methanotroph Methylocystis parvus OBBP, which, unlike Methylocystis sp. strain M, does not possess an sMMO. A similar phylogenetic analysis using the pmoA gene, which encodes the 27-kDa polypeptide of the particulate MMO, also places Methylocystis sp. strain M firmly in the genus Methylocystis. This is the first report of isolation and characterization of methane oxidation genes from methanotrophs of the genus Methylocystis.