Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Jan 15;369(Pt 2):417–427. doi: 10.1042/BJ20020823

The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein.

Piku Basu 1, Bettina Katterle 1, K Kristoffer Andersson 1, Howard Dalton 1
PMCID: PMC1223091  PMID: 12379148

Abstract

A protocol has been developed which permits the purification of a membrane-associated methane-oxidizing complex from Methylococcus capsulatus (Bath). This complex has approximately 5 fold higher specific activity than any purified particulate methane mono-oxygenase (pMMO) previously reported from M. capsulatus (Bath). This efficiently functioning methane-oxidizing complex consists of the pMMO hydroxylase (pMMOH) and an unidentified component we have assigned as a potential pMMO reductase (pMMOR). The complex was isolated by solubilizing intracytoplasmic membrane preparations containing the high yields of active membrane-bound pMMO (pMMO(m)), using the non-ionic detergent dodecyl-beta-D-maltoside, to yield solubilized enzyme (pMMO(s)). Further purification gave rise to an active complex (pMMO(c)) that could be resolved (at low levels) by ion-exchange chromatography into two components, the pMMOH (47, 27 and 24 kDa subunits) and the pMMOR (63 and 8 kDa subunits). The purified complex contains two copper atoms and one non-haem iron atom/mol of enzyme. EPR spectra of preparations grown with (63)Cu indicated that the copper ion interacted with three or four nitrogenic ligands. These EPR data, in conjunction with other experimental results, including the oxidation by ferricyanide, EDTA treatment to remove copper and re-addition of copper to the depleted protein, verified the essential role of copper in enzyme catalysis and indicated the implausibility of copper existing as a trinuclear cluster. The EPR measurements also demonstrated the presence of a tightly bound mononuclear Fe(3+) ion in an octahedral environment that may well be exchange-coupled to another paramagnetic species.

Full Text

The Full Text of this article is available as a PDF (198.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Brusseau G. A., Tsien H. C., Hanson R. S., Wackett L. P. Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity. Biodegradation. 1990;1(1):19–29. doi: 10.1007/BF00117048. [DOI] [PubMed] [Google Scholar]
  3. Fox B. G., Froland W. A., Dege J. E., Lipscomb J. D. Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph. J Biol Chem. 1989 Jun 15;264(17):10023–10033. [PubMed] [Google Scholar]
  4. Green J., Dalton H. Protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath). A novel regulatory protein of enzyme activity. J Biol Chem. 1985 Dec 15;260(29):15795–15801. [PubMed] [Google Scholar]
  5. Green J., Dalton H. The biosynthesis and assembly of protein A of soluble methane monooxygenase of Methylococcus capsulatus (Bath). J Biol Chem. 1988 Nov 25;263(33):17561–17565. [PubMed] [Google Scholar]
  6. Katterle Bettina, Gvozdev Rudolf I., Abudu Ntei, Ljones Torbjørn, Andersson K. Kristoffer. A continuous-wave electron-nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine beta-mono-oxygenase of the adrenal medulla. Biochem J. 2002 May 1;363(Pt 3):677–686. doi: 10.1042/0264-6021:3630677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Lemos S. S., Perille Collins M. L., Eaton S. S., Eaton G. R., Antholine W. E. Comparison of EPR-visible Cu(2+) sites in pMMO from Methylococcus capsulatus (Bath) and Methylomicrobium album BG8. Biophys J. 2000 Aug;79(2):1085–1094. doi: 10.1016/s0006-3495(00)76362-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Liu K. E., Lippard S. J. Redox properties of the hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath). Effects of protein B, reductase, and substrate. J Biol Chem. 1991 Jul 15;266(20):12836–12839. [PubMed] [Google Scholar]
  10. Lo C. M., Fan S. T., Liu C. L., Lo R. J., Lai C. L., Lau G. K., Chan J. K., Ng I. O., Wong J. Five-year experience with the development of a liver transplant program in Hong Kong. Transplant Proc. 1998 Nov;30(7):3247–3248. doi: 10.1016/s0041-1345(98)01013-6. [DOI] [PubMed] [Google Scholar]
  11. Lund J., Dalton H. Further characterisation of the FAD and Fe2S2 redox centres of component C, the NADH:acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath). Eur J Biochem. 1985 Mar 1;147(2):291–296. doi: 10.1111/j.1432-1033.1985.tb08749.x. [DOI] [PubMed] [Google Scholar]
  12. MacMillan F., Kannt A., Behr J., Prisner T., Michel H. Direct evidence for a tyrosine radical in the reaction of cytochrome c oxidase with hydrogen peroxide. Biochemistry. 1999 Jul 20;38(29):9179–9184. doi: 10.1021/bi9911987. [DOI] [PubMed] [Google Scholar]
  13. Nguyen H. H., Elliott S. J., Yip J. H., Chan S. I. The particulate methane monooxygenase from methylococcus capsulatus (Bath) is a novel copper-containing three-subunit enzyme. Isolation and characterization. J Biol Chem. 1998 Apr 3;273(14):7957–7966. doi: 10.1074/jbc.273.14.7957. [DOI] [PubMed] [Google Scholar]
  14. Nguyen H. H., Shiemke A. K., Jacobs S. J., Hales B. J., Lidstrom M. E., Chan S. I. The nature of the copper ions in the membranes containing the particulate methane monooxygenase from Methylococcus capsulatus (Bath). J Biol Chem. 1994 May 27;269(21):14995–15005. [PubMed] [Google Scholar]
  15. Que Lawrence, Jr, Ho Raymond Y. N. Dioxygen Activation by Enzymes with Mononuclear Non-Heme Iron Active Sites. Chem Rev. 1996 Nov 7;96(7):2607–2624. doi: 10.1021/cr960039f. [DOI] [PubMed] [Google Scholar]
  16. Sahlin M., Petersson L., Gräslund A., Ehrenberg A., Sjöberg B. M., Thelander L. Magnetic interaction between the tyrosyl free radical and the antiferromagnetically coupled iron center in ribonucleotide reductase. Biochemistry. 1987 Aug 25;26(17):5541–5548. doi: 10.1021/bi00391a049. [DOI] [PubMed] [Google Scholar]
  17. Shiemke A. K., Cook S. A., Miley T., Singleton P. Detergent solubilization of membrane-bound methane monooxygenase requires plastoquinol analogs as electron donors. Arch Biochem Biophys. 1995 Aug 20;321(2):421–428. doi: 10.1006/abbi.1995.1413. [DOI] [PubMed] [Google Scholar]
  18. Smith D. D., Dalton H. Solubilisation of methane monooxygenase from Methylococcus capsulatus (Bath). Eur J Biochem. 1989 Jul 1;182(3):667–671. doi: 10.1111/j.1432-1033.1989.tb14877.x. [DOI] [PubMed] [Google Scholar]
  19. Takeguchi M., Miyakawa K., Okura I. Properties of the membranes containing the particulate methane monooxygenase from Methylosinus trichosporium OB3b. Biometals. 1998 Sep;11(3):229–234. doi: 10.1023/a:1009278216452. [DOI] [PubMed] [Google Scholar]
  20. Tate S., Dalton H. A low-molecular-mass protein from Methylococcus capsulatus (Bath) is responsible for the regulation of formaldehyde dehydrogenase activity in vitro. Microbiology. 1999 Jan;145(Pt 1):159–167. doi: 10.1099/13500872-145-1-159. [DOI] [PubMed] [Google Scholar]
  21. Tonge G. M., Harrison D. E., Higgins I. J. Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b. Biochem J. 1977 Feb 1;161(2):333–344. doi: 10.1042/bj1610333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tonge G. M., Harrison D. E., Knowles C. J., Higgins I. J. Properties and partial purification of the methane-oxidising enzyme system from Methylosinus trichosporium. FEBS Lett. 1975 Oct 15;58(1):293–299. doi: 10.1016/0014-5793(75)80282-1. [DOI] [PubMed] [Google Scholar]
  23. Wallar Bradley J., Lipscomb John D. Dioxygen Activation by Enzymes Containing Binuclear Non-Heme Iron Clusters. Chem Rev. 1996 Nov 7;96(7):2625–2658. doi: 10.1021/cr9500489. [DOI] [PubMed] [Google Scholar]
  24. Whittenbury R., Phillips K. C., Wilkinson J. F. Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol. 1970 May;61(2):205–218. doi: 10.1099/00221287-61-2-205. [DOI] [PubMed] [Google Scholar]
  25. Yuan H., Collins M. L., Antholine W. E. Concentration of Cu, EPR-detectable Cu, and formation of cupric-ferrocyanide in membranes with pMMO. J Inorg Biochem. 1998 Dec;72(3-4):179–185. doi: 10.1016/s0162-0134(98)10078-8. [DOI] [PubMed] [Google Scholar]
  26. Yuan H., Collins M. L., Antholine W. E. Type 2 Cu2+ in pMMO from Methylomicrobium album BG8. Biophys J. 1999 Apr;76(4):2223–2229. doi: 10.1016/S0006-3495(99)77378-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zahn J. A., Arciero D. M., Hooper A. B., DiSpirito A. A. Evidence for an iron center in the ammonia monooxygenase from Nitrosomonas europaea. FEBS Lett. 1996 Nov 11;397(1):35–38. doi: 10.1016/s0014-5793(96)01116-7. [DOI] [PubMed] [Google Scholar]
  28. Zahn J. A., DiSpirito A. A. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath). J Bacteriol. 1996 Feb;178(4):1018–1029. doi: 10.1128/jb.178.4.1018-1029.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES