Vol. 126, No. 2 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, May 1976, p. 1017-1019 Copyright C 1976 American Society for Microbiology

Inhibition of Dimethyl Ether and Methane Oxidation in Methylococcus capsulatus and Methylosinus trichosporium RAMESH PATEL,* C. T. HOU, AND A. FELIX Corporate Research Laboratories, EXXON Research and Engineering Company, Linden, New Jersey 07036 Received for publication 19 January 1976

Metal-chelating or -binding agents inhibited the oxidation of dimethyl ether and methane, but not methanol, by cell suspensions of Methylococcus capsulatus and Methylosinus trichosporium. Evidence suggests the involvement of metal-containing enzymatic systems in the initial step of oxidation of dimethyl ether and methane. The methane-oxidizing bacteria are obligately dependent on methane, methanol, or dimethyl ether as sources of carbon and energy for growth (6, 16, 17). Recently, Patt et al. (12) isolated facultative methane-oxidizing bacteria that will utilize methane as well as the more complex organic carbon and energy sources. All methane-oxidizing bacteria so far described possess extensive intracytoplasmic membrane (4, 12, 15). On the basis of structural organization of their intracytoplasmic membrane and pathway of carbon assimilation, the methaneoxidizing bacteria are divided into two distinct groups (13, 15). Cell suspensions of methanegrown organisms oxidize methane, dimethyl ether, methanol, formaldehyde, and formate (1, 9, 11). Involvement of monooxygenase in methane oxidation has been implicated by methanestimulated reduced nicotinamide adenine dinucleotide oxidation or oxygen consumption observed in cell-free particulate fractions ofMethylococcus capsulatus (14) and Pseudomonas methanica (5). Recently, Hubley et al. (7) demonstrated that the oxidation of methane by cell suspensions of Methylosinus trichosporium was inhibited by metal-binding compounds. They suggested a role for copper in the methane oxygenase on the basis of the pattern of inhibition and relief of inhibition by added metal ions. In this communication, we report the inhibition by various compounds of dimethyl ether and methane oxidation in Methylococcus capsulatus TRMC and Methylosinus trichosporium OB3b. The organisms selected for study represent two distinct groups of methane-oxidizing bacteria (13, 15). M. capsulatus TRMC and M. trichosporium OB3b were grown in 2.8-liter flasks containing 700 ml of mineral salts medium (6) in an atmosphere of methane and air (50:50, vol/vol) at

30 C. Cells were harvested after 40 h in the exponential phase of growth by centrifuging for 15 min at 12,000 x g and were washed twice in 25 mM potassium phosphate buffer, pH 7.0. Cells were suspended in the same buffer. Respiration rates were measured at 28 C by oxygen consumption with a Clark oxygen electrode (Yellow Springs Instruments Co., Yellow Springs, Ohio). Reaction mixtures contained, in a final volume of 3 ml: dimethyl ether- or methane-saturated 25 mM potassium phosphate buffer, pH 7.0 (0.5 ril); air-saturated 25 mM potassium phosphate buffer, pH 7.0; and an appropriate amount of inhibitor. The reaction was started by injecting 50 ,ul of cell suspensions, and the rate of oxygen consumption was measured. Protein in the cell suspension was estimated by the method of Lowry et al. (10), using bovine serum albumin as standard. The oxidation of dimethyl ether and methane by cell suspensions of M. capsulatus TRMC and M. trichosporium OB3b was inhibited by various metal-binding compounds with different ligand combinations, i.e., nitrogen-nitrogen (a,a-bipyridyl), oxygen-nitrogen (8-hydroxyquinoline), and sulfur-nitrogen (thiourea, thiosemicarbazide) (Tables 1 and 2). This suggests the involvement of metal ion(s) in the oxidation of both dimethyl ether and methane. Oxidation of methanol was not inhibited under similar conditions, which suggests that inhibitors act on the initial step in the metabolism of dimethyl ether and methane. Since CO is known to interact with monooxygenases associated with cytochrome P-450, which is involved in many bacterial monooxygenase reactions (2, 8), the effect of CO on the oxidation of dimethyl ether and methane was tested. Oxidation of both compounds was inhibited by CO (Tables 1 and 2). Davey and Milton (3) have discussed the participation of

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TABLE 1. Inhibition of dimethyl ether and methane oxidation in cell suspensions of Methylococcus capsulatus TRMCa Inhibition (%)

(M)c Inhibitor I r(M)

Di-

methyl

Methane

ether

100 100 Thiourea ........... 10-3 72 80 10-3 1,10-Phenanthroline 100 75 10-3 8-Hydroxyquinoline 100 70 a, a-Bipyridyl ....... 10-3 80 50 10-3 Thiosemicarbazide .. 100 100 Imidazole ........... 10-3 100 80 Potassium cyanide . . 10-4 80 64 Carbon monoxide .... NDb 40 30 Acriflavin .......... 10-3 60 45 Antimycin A ........ 10-3 a The uninhibited rates of dimethyl ether and methane oxidations were 460 and 520 nmol of oxygen consumed/min per mg of protein, respectively. b Not done.

TABLE 2. Inhibition of dimethyl ether and methane oxidation in cell suspensions of Methylosinus trichosporium OB3ba Inhibition (%)

Inhibitor

Concn

(M)

Dimethyl ether

Methane

100 100 Thiourea ........... 10-3 84 92 10-3 1,10-Phenanthroline 10-3 100 88 8-Hydroxyquinoline 90 75 a,a-Bipyridyl ... 10-3 10-3 80 100 Thiosemicarbazide .. 80 90 Imidazole ........... 10-3 100 75 Potassium cyanide .. 10-4 89 53 Carbon monoxide .... NDb 20 36 Acriflavin .......... 10-3 68 40 Antimycin A ....... 10-3 a The uninhibited rates of dimethyl ether and methane oxidation were 400 and 480 nmol of oxygen consumed/min per mg of protein, respectively. b Not done.

CO-binding cytochrome in methane oxidation. Inhibition of dimethyl ether oxidation by CO may be due to a similar mechanism. Another interpretation concerning the mechanism of CO inhibition came when it was reported that methane and CO oxidation are catalyzed by a single enzyme system (5). This leads to the possibility of an inhibitory effect of CO on methane oxidation by competition for the same enzyme system. It is possible that inhibition of dimethyl ether oxidation by CO may be due to nonspecificity of the methane oxygenase system, which may catalyze dimethyl ether oxidation. Acriflavin and antimycin A, inhibitors of electron transport, only partially inhibited oxi-

dation of dimethyl ether and methane in both organisms. Recently, Ribbons (14) reported that methane-dependent reduced nicotinamide adenine dinucleotide oxidase activity in a particulate fraction of M. capsulatus was inhibited by various inhibitors of the electron transport system. He has also reported oxidation of dimethyl ether by the particulate fraction from M. capsulatus. Quayle (13) has proposed two different mechanisms for the conversion of dimethyl ether to methanol by methane-oxidizing bacteria. One may be due to the reaction catalyzed by a monooxygenase system: CH3-0-CH3 + XH2 + 02 -+ CH30H + HCHO + X + H20

Another may be a cleavage reaction as suggested initially by Bryan Jones and Wilkinson

(17): CH3-0-CH3 + XH2

-*

CH30H + CH3X

In conclusion, inhibition of dimethyl ether and methane oxidation by various inhibitors suggests the possibility of the involvement of a metal-containing analogous enzymatic system(s) in the initial step of oxidation of these compounds by both types of methane-oxidizing bacteria. LITERATURE CITED 1. Brown, L. R., R. J. Strawinsky, and C.S. McClesky. 1964. The isolation and characterization of Methanomonas methanooxidans. Can. J. Microbiol. 10:791-

799. 2. Cardini, G., and P. Jurtshuk. 1968. Cytochrome P-450 involvement in oxidation of n-octane by cell-free extract of Corynebacterium. J. Biol. Chem. 243:60706072. 3. Davey, J. F., and J. R. Milton. 1973. Cytochrome of two methane-utilizing bacteria. FEBS Lett. 37:335-337. 4. Davies, S. L., and R. Whittenbury. 1970. Fine structure of methane-utilizing bacteria. J. Gen. Microbiol. 61:227-232. 5. Ferenci, T. 1974. Carbon monoxide-stimulated respiration in methane-utilizing bacteria. FEBS Lett. 41:9498. 6. Foster, J. W., and R. H. Davies. 1966. A methanedependent coccus, with notes on classification and nomenclature of obligate methane-utilizing bacteria. J. Bacteriol. 91:1924-1931. 7. Hubley, J. H., A. W. Thomson, and J. F. Wilkinson. 1975. Specific inhibitors of methane oxidation in

Methylsinus trichosporium. Arch. Microbiol. 102:199202. 8. Katagiri, M., B. N. Ganguli, and I. C. Gunsalus. 1968. Soluble cytochrome P-450 functional in methylene hydroxylation. J. Biol. Chem. 243:3543-3546. 9. Leadbetter, E. R., and J. W. Foster. 1958. Studies on some methane-utilizing bacteria. Arch. Mikrobiol. 30:51-118. 10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 11. Patel, R. N., and D. S. Hoare. 1971. Physiological stud-

VOL. 126, 1976 ies of methane- and methanol-oxidizing bacteria: oxidation of C-1 compounds by Methylococcus capsulatus. J. Bacteriol. 107:187-192. 12. Patt, T. E., G. C. Cole, J. Bland, and R. S. Hanson. 1974. Isolation and characterization of bacteria that grow on methane and organic compounds as sole sources of carbon and energy. J. Bacteriol. 120:955964. 13. Quayle, J. R. 1972. The metabolism of c-1 compounds by micro-organisms, p. 119-203. In A. H. Rose and D. W. Tempest (ed.), Advances in microbial physiology, vol. 7. Academic Press Inc., London. 14. Ribbons, D. W. 1975. Oxidation of c-1 compounds by

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particulate fractions from Methylococcus capsulatus: distribution and properties of methane-dependent reduced nicotinamide adenine dinucleotide oxidase (methane hydroxylase). J. Bacteriol. 122:1351-1363. 15. Ribbons, D. W., J. E. Harrison, and A. M. Wadzinski. 1970. Metabolism of single carbon compounds. Annu. Rev. Microbiol. 24:135-138. 16. Whittenbury, R., K. C. Phillips, and J. F. Wilkinson. 1970. Enrichment, isolation and some properties of methane-utilizing bacteria. J. Gen. Microbiol. 61:205-218. 17. Wilkinson, J. F. 1971. Hydrocarbon

as a source of single-cell protein. Symp. Soc. Gen. Microbiol. 21:15-46.

Inhibition of dimethyl ether and methane oxidation in Methylococcus capsulatus and Methylosinus trichosporium.

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