The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme
1998; Elsevier BV; Volume: 273; Issue: 14 Linguagem: Inglês
10.1074/jbc.273.14.7957
ISSN1083-351X
AutoresHiep-Hoa T. Nguyen, Sean J. Elliott, John H. K. Yip, Sunney I. Chan,
Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoThe particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro. By cultivatingMethylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells. Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl β-d-maltoside and purification by chromatography. The pMMO was found to be the major constituent in these membranes, constituting 60–80% of total membrane proteins. The dominant species of the pMMO was found to consist of three subunits, α, β, and γ, with an apparent molecular mass of 45, 26, and 23 kDa, respectively. A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, α′, β, and γ, with an apparent molecular mass of 35, 26, and 23 kDa, respectively. The α and α′ subunits from these two forms of the pMMO contain identical N-terminal sequences. The γ subunit, however, exhibits variation in its N-terminal sequence. The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions. Approximately 12–15 Cu ions per 94-kDa monomeric unit were observed. The pMMO is sensitive to dioxygen tension. On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished. A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO. This form was the subject of our pMMO isolation effort. Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure. Under inactivating conditions, these unstable forms release protons to the buffer (∼10 H+/94-kDa monomeric unit) and eventually become completely inactive. The particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro. By cultivatingMethylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells. Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl β-d-maltoside and purification by chromatography. The pMMO was found to be the major constituent in these membranes, constituting 60–80% of total membrane proteins. The dominant species of the pMMO was found to consist of three subunits, α, β, and γ, with an apparent molecular mass of 45, 26, and 23 kDa, respectively. A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, α′, β, and γ, with an apparent molecular mass of 35, 26, and 23 kDa, respectively. The α and α′ subunits from these two forms of the pMMO contain identical N-terminal sequences. The γ subunit, however, exhibits variation in its N-terminal sequence. The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions. Approximately 12–15 Cu ions per 94-kDa monomeric unit were observed. The pMMO is sensitive to dioxygen tension. On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished. A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO. This form was the subject of our pMMO isolation effort. Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure. Under inactivating conditions, these unstable forms release protons to the buffer (∼10 H+/94-kDa monomeric unit) and eventually become completely inactive. The enzyme methane monooxygenase, found in methanotrophic bacteria, catalyzes the conversion of methane to methanol using dioxygen as a co-substrate at ambient temperatures and pressures (1Anthony C. The Biochemistry of Methylotrophs. Academic Press, London1982: 296-379Google Scholar,2Bédard C. Knowles R. Microbiol. Rev. 1989; 53: 68-84Crossref PubMed Google Scholar). This system has attracted considerable attention, since it provides an ideal natural model to study methane activation and functionalization, a subject of significant current interest (3Crabtree R.H. Chem. Rev. 1995; 95: 987-1007Crossref Scopus (793) Google Scholar). Two distinct species of methane monooxygenase (MMO) 1The abbreviations used are: MMO, methane monooxygenase; sMMO, soluble methane monooxygenase; pMMO, particulate methane monooxygenase; pMMOH, pMMO hydroxylase; AMO, ammonia monooxygenase; Pipes, piperazine-N,N′-bis(2-ethanesulfonic acid); PAGE, polyacrylamide gel electrophoresis. are known to exist at different cellular locations, a cytoplasmic (soluble) MMO and a membrane-bound (particulate) MMO (4Lipscomb J.D. Annu. Rev. Microbiol. 1994; 48: 371-399Crossref PubMed Scopus (339) Google Scholar). The soluble MMO (sMMO) is a complex three-component system consisting of a hydroxylase, a reductase, and a small regulatory protein (4Lipscomb J.D. Annu. Rev. Microbiol. 1994; 48: 371-399Crossref PubMed Scopus (339) Google Scholar). The sMMO has been investigated extensively by several research groups (5Fox B.G. Froland W.A. Jollie D.R. Lipscomb J.D. 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Chem. 1994; 269: 14995-15005Abstract Full Text PDF PubMed Google Scholar, 25Nguyen H.-H.N.T. Zhu M. Elliott S.J. Nakagawa K.H. Hedman B. Costello A.M. Peeples T.L. Wilkinson B. Morimoto H. Williams P.G. Floss H.G. Lidstrom M.E. Hodgson K.O. Chan S.I. Lidstrom M.E. Tabita F.R. Microbial Growth on C1 Compounds. Kluwer Academic Publishers, Norwell, MA1996: 150-158Crossref Google Scholar, 26Nguyen H.-H.T. Nakagawa K.H. Hedman B. Elliott S.J. Lidstrom M.E. Hodgson K.O. Chan S.I. J. Am. Chem. Soc. 1996; 118: 12766-12776Crossref Scopus (114) Google Scholar, 27Semrau J.D. Zolandz D. Lidstrom M.E. Chan S.I. J. Inorg. Biochem. 1995; 58: 235-244Crossref PubMed Scopus (60) Google Scholar, 28Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Karlin K.D. Tyeklar Z. Bioinorganic Chemistry of Copper. Chapman and Hall, New York1993: 184-195Crossref Google Scholar, 29Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Murrell J.C. Kelly D.P. Microbial Growth on C1 Compounds. 1993: 93-107Google Scholar). This enzyme is much less well characterized mainly due to its unusual activity instability. Despite the lability of the enzyme activity in vitro, the pMMO appears to be expressed in all methanotrophs (1Anthony C. The Biochemistry of Methylotrophs. Academic Press, London1982: 296-379Google Scholar, 2Bédard C. Knowles R. Microbiol. Rev. 1989; 53: 68-84Crossref PubMed Google Scholar, 4Lipscomb J.D. Annu. Rev. Microbiol. 1994; 48: 371-399Crossref PubMed Scopus (339) Google Scholar). So far, the sMMO has been detected in only the following strains and species:M. capsulatus, Methylosinus trichosporium, Methylosinus sporium, Methylocystis sp. M and Methylomonas methanica 68–1 (6Pilkington S.J. Dalton H. Methods Enzymol. 1990; 188: 181-190Crossref Scopus (63) Google Scholar, 30Stainthorpe A.C. Salmond G.P.C. Dalton H. Murrell J.C. FEMS Microbiol. Lett. 1990; 70: 103-108Crossref Scopus (33) Google Scholar, 31Stainthorpe A.C. 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Lett. 1983; 5: 487-492Crossref Scopus (316) Google Scholar, 38Scott D. Brannan J. Higgins I.J. J. Gen. Microbiol. 1981; 125: 63-72Google Scholar, 39Dalton, H., Prior, S. D., Leak, D. J., Stanley, S. J. H., Microbial Growth on C1 Compounds, Crawford, R. L., Hanson, R. S., 1984, 75, 82, ., Intercept, Andover, Hampshire, United Kingdom.Google Scholar). Otherwise, the pMMO is expressed. Copper ions not only regulate the expression of the pMMO but have been found to be crucial for pMMO activity. The expression of the pMMO is accompanied by the formation of an extensive network of intracytoplasmic membranes, where the membrane-bound pMMO resides (35Cornish A. MacDonald J. Burrows K.J. King T.S. Scott D. Higgins I.J. Biotechnol. Lett. 1985; 5: 319-324Crossref Scopus (20) Google Scholar, 36Prior S.D. Dalton H. J. Gen. Microbiol. 1985; 131: 155-163Google Scholar, 37Stanley S.H. Prior S.D. Leak D.J. Dalton H. Biotechnol. Lett. 1983; 5: 487-492Crossref Scopus (316) Google Scholar, 38Scott D. Brannan J. Higgins I.J. J. Gen. Microbiol. 1981; 125: 63-72Google Scholar, 39Dalton, H., Prior, S. D., Leak, D. J., Stanley, S. J. H., Microbial Growth on C1 Compounds, Crawford, R. L., Hanson, R. S., 1984, 75, 82, ., Intercept, Andover, Hampshire, United Kingdom.Google Scholar). An increase in carbon to biomass conversion efficiency is also observed. Three new polypeptides with apparent molecular masses of 45, 35, and 26 kDa were observed in the membrane fractions when M. capsulatus (Bath) switched from expressing the sMMO to the pMMO (35Cornish A. MacDonald J. Burrows K.J. King T.S. Scott D. Higgins I.J. Biotechnol. Lett. 1985; 5: 319-324Crossref Scopus (20) Google Scholar, 36Prior S.D. Dalton H. J. Gen. Microbiol. 1985; 131: 155-163Google Scholar, 37Stanley S.H. Prior S.D. Leak D.J. Dalton H. Biotechnol. Lett. 1983; 5: 487-492Crossref Scopus (316) Google Scholar, 38Scott D. Brannan J. Higgins I.J. J. Gen. Microbiol. 1981; 125: 63-72Google Scholar, 39Dalton, H., Prior, S. D., Leak, D. J., Stanley, S. J. H., Microbial Growth on C1 Compounds, Crawford, R. L., Hanson, R. S., 1984, 75, 82, ., Intercept, Andover, Hampshire, United Kingdom.Google Scholar). Recent progress in our laboratory indicates that the pMMO is a novel copper-containing enzyme. Metal/protein ratio data analysis clearly suggests that the pMMO is a multiple copper-containing enzyme (24Nguyen H.-H.T. Shiemke A.K. Jacobs S.J. Hales B.J. Lidstrom M.E. Chan S.I. J. Biol. Chem. 1994; 269: 14995-15005Abstract Full Text PDF PubMed Google Scholar, 25Nguyen H.-H.N.T. Zhu M. Elliott S.J. Nakagawa K.H. Hedman B. Costello A.M. Peeples T.L. Wilkinson B. Morimoto H. Williams P.G. Floss H.G. Lidstrom M.E. Hodgson K.O. Chan S.I. Lidstrom M.E. Tabita F.R. Microbial Growth on C1 Compounds. Kluwer Academic Publishers, Norwell, MA1996: 150-158Crossref Google Scholar, 26Nguyen H.-H.T. Nakagawa K.H. Hedman B. Elliott S.J. Lidstrom M.E. Hodgson K.O. Chan S.I. J. Am. Chem. Soc. 1996; 118: 12766-12776Crossref Scopus (114) Google Scholar,28Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Karlin K.D. Tyeklar Z. Bioinorganic Chemistry of Copper. Chapman and Hall, New York1993: 184-195Crossref Google Scholar, 29Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Murrell J.C. Kelly D.P. Microbial Growth on C1 Compounds. 1993: 93-107Google Scholar, 40Semrau J.D. Chistoserdov A. Lebron J. Costello A. Davagnino J. Kenna E. Holmes A.J. Finch R. Murrell J.C. Lidstrom M.E. J. Bacteriol. 1995; 177: 3071-3079Crossref PubMed Google Scholar). Activity was found to be proportional to the level of membrane-bound copper ions (24Nguyen H.-H.T. Shiemke A.K. Jacobs S.J. Hales B.J. Lidstrom M.E. Chan S.I. J. Biol. Chem. 1994; 269: 14995-15005Abstract Full Text PDF PubMed Google Scholar, 27Semrau J.D. Zolandz D. Lidstrom M.E. Chan S.I. J. Inorg. Biochem. 1995; 58: 235-244Crossref PubMed Scopus (60) Google Scholar, 28Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Karlin K.D. Tyeklar Z. Bioinorganic Chemistry of Copper. Chapman and Hall, New York1993: 184-195Crossref Google Scholar, 29Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Murrell J.C. Kelly D.P. Microbial Growth on C1 Compounds. 1993: 93-107Google Scholar). The pMMO-associated copper ions appear to be organized into trinuclear cluster units with rather defined magnetic and redox properties (24Nguyen H.-H.T. Shiemke A.K. Jacobs S.J. Hales B.J. Lidstrom M.E. Chan S.I. J. Biol. Chem. 1994; 269: 14995-15005Abstract Full Text PDF PubMed Google Scholar, 25Nguyen H.-H.N.T. Zhu M. Elliott S.J. Nakagawa K.H. Hedman B. Costello A.M. Peeples T.L. Wilkinson B. Morimoto H. Williams P.G. Floss H.G. Lidstrom M.E. Hodgson K.O. Chan S.I. Lidstrom M.E. Tabita F.R. Microbial Growth on C1 Compounds. Kluwer Academic Publishers, Norwell, MA1996: 150-158Crossref Google Scholar, 26Nguyen H.-H.T. Nakagawa K.H. Hedman B. Elliott S.J. Lidstrom M.E. Hodgson K.O. Chan S.I. J. Am. Chem. Soc. 1996; 118: 12766-12776Crossref Scopus (114) Google Scholar, 28Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Karlin K.D. Tyeklar Z. Bioinorganic Chemistry of Copper. Chapman and Hall, New York1993: 184-195Crossref Google Scholar, 29Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Murrell J.C. Kelly D.P. Microbial Growth on C1 Compounds. 1993: 93-107Google Scholar, 40Semrau J.D. Chistoserdov A. Lebron J. Costello A. Davagnino J. Kenna E. Holmes A.J. Finch R. Murrell J.C. Lidstrom M.E. J. Bacteriol. 1995; 177: 3071-3079Crossref PubMed Google Scholar). The as-isolated pMMO-enriched membranes often contain a mixture of Cu(I) and Cu(II) ions in various proportions, depending on the handling of the samples (25Nguyen H.-H.N.T. Zhu M. Elliott S.J. Nakagawa K.H. Hedman B. Costello A.M. Peeples T.L. Wilkinson B. Morimoto H. Williams P.G. Floss H.G. Lidstrom M.E. Hodgson K.O. Chan S.I. Lidstrom M.E. Tabita F.R. Microbial Growth on C1 Compounds. Kluwer Academic Publishers, Norwell, MA1996: 150-158Crossref Google Scholar, 26Nguyen H.-H.T. Nakagawa K.H. Hedman B. Elliott S.J. Lidstrom M.E. Hodgson K.O. Chan S.I. J. Am. Chem. Soc. 1996; 118: 12766-12776Crossref Scopus (114) Google Scholar, 28Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Karlin K.D. Tyeklar Z. Bioinorganic Chemistry of Copper. Chapman and Hall, New York1993: 184-195Crossref Google Scholar, 29Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Murrell J.C. Kelly D.P. Microbial Growth on C1 Compounds. 1993: 93-107Google Scholar, 40Semrau J.D. Chistoserdov A. Lebron J. Costello A. Davagnino J. Kenna E. Holmes A.J. Finch R. Murrell J.C. Lidstrom M.E. J. Bacteriol. 1995; 177: 3071-3079Crossref PubMed Google Scholar). Hence, the functional form of the enzyme has been suggested to be the reduced or partially reduced form. The chemistry catalyzed by this enzyme is also highly specific. pMMO-catalyzed hydroxylation of cryptically chiral ethanes has implicated a reaction mechanism proceeding with complete retention of alkane substrate configuration (41Priestly N.D. Floss H.G. Froland W.A. Lipscomb J.D. Williams P.G. Morimoto H. J. Am. Chem. Soc. 1992; 114: 7561-7562Crossref Scopus (153) Google Scholar, 42Wilkinson B. Zhu M. Priestley N.D. Nguyen H.-H.T. Morimoto H. Williams P.G. Chan S.I. Floss H.G. J. Am. Chem. Soc. 1996; 118: 921-922Crossref Scopus (89) Google Scholar). This extraordinary chemistry currently has no precedent in known model and biological systems. Accordingly, insights regarding the copper-containing active site of the pMMO can provide a new direction in the design of biominetic catalysts for methane activation and functionalization. The pMMO has been known to be very difficult to study. As noted earlier, one of the main obstacles in studying the pMMO is the unusual instability of the activity of the enzyme. Activity is frequently lost upon cell lysis, detergent solubilization, and freeze-thaw cycles. In several cultures, no activity was observed in cell-free extracts, or activity quickly disappeared within 6 h after cell lysis (24Nguyen H.-H.T. Shiemke A.K. Jacobs S.J. Hales B.J. Lidstrom M.E. Chan S.I. J. Biol. Chem. 1994; 269: 14995-15005Abstract Full Text PDF PubMed Google Scholar, 25Nguyen H.-H.N.T. Zhu M. Elliott S.J. Nakagawa K.H. Hedman B. Costello A.M. Peeples T.L. Wilkinson B. Morimoto H. Williams P.G. Floss H.G. Lidstrom M.E. Hodgson K.O. Chan S.I. Lidstrom M.E. Tabita F.R. Microbial Growth on C1 Compounds. Kluwer Academic Publishers, Norwell, MA1996: 150-158Crossref Google Scholar, 26Nguyen H.-H.T. Nakagawa K.H. Hedman B. Elliott S.J. Lidstrom M.E. Hodgson K.O. Chan S.I. J. Am. Chem. Soc. 1996; 118: 12766-12776Crossref Scopus (114) Google Scholar,28Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Karlin K.D. Tyeklar Z. Bioinorganic Chemistry of Copper. Chapman and Hall, New York1993: 184-195Crossref Google Scholar, 29Chan S.I. Nguyen H.-H.T. Shiemke A.K. Lidstrom M.E. Murrell J.C. Kelly D.P. Microbial Growth on C1 Compounds. 1993: 93-107Google Scholar, 40Semrau J.D. Chistoserdov A. Lebron J. Costello A. Davagnino J. Kenna E. Holmes A.J. Finch R. Murrell J.C. Lidstrom M.E. J. Bacteriol. 1995; 177: 3071-3079Crossref PubMed Google Scholar). Enzymatic activity is also known to be very sensitive to exogenous ligands as well as the choice of buffer. This highly unusual instability has hampered efforts in characterizing the pMMO. The addition of copper ions is known to enhance enzymatic activity under certain conditions (cells grown at low copper levels), but the effect of copper ions in the extension of pMMO activity is not known. No reagent is currently known to reactivate the enzyme once the protein becomes inactive. As a result, a highly active, stable, and purified preparation of the enzyme has been slow in forthcoming. Past efforts in isolating the pMMO have resulted in significant confusion regarding the nature of the enzyme. An early report of pMMO isolation from M. trichosporium OB3b indicates that this system can utilize ascorbate in addition to NADH as electron donors and was found to consist of three components: a 47-kDa polypeptide containing various amounts of copper, a smaller 9.4-kDa subunit, and a 13-kDa CO-binding cytochrome c (43Tonge G.M. Harrison D.E.F. Higgins I.J. Biochem. J. 1977; 161: 333-344Crossref PubMed Scopus (83) Google Scholar). In later studies, the aforementioned ascorbate-linked activity was not observed, and attempts to solubilize the enzyme resulted in complete deactivation of the protein. Solubilization of pMMO from M. capsulatus (Bath) using a nonionic detergent was attempted, and upon detergent removal and lipid vesicle reconstitution, partial activity was observed (44Drummond D. Smith D. Dalton H. Eur. J. Biochem. 1989; 1982: 667-671Google Scholar). According to the authors, any attempts to purify the enzyme further resulted in complete loss of activity. A few reports including a recent work (45Zahn J.A. DiSpirito A.A. J. Bacteriol. 1996; 178: 1018-1029Crossref PubMed Google Scholar, 46Akent'eva N.F. Gvozdev R.I. Biochemistry (Engl. Transl. Biokhimiya). 1988; 53: 91-96Google Scholar) appear to support the notion that the active site of the pMMO may contain iron despite the overwhelming evidence accumulated to date suggesting that copper is the element responsible for catalysis within the enzyme active site. Thus, to advance the field, the presence of iron and copper in the pMMO must be resolved. This paper summarizes our efforts to isolate and purify the pMMO fromM. capsulatus (Bath) for biochemical and biophysical characterization. Toward the development of suitable protocols for pMMO isolation, we have embarked on an extensive investigation of factors contributing to enzymatic activity stability, including various methods of bacterial cultivation and membrane isolation, and various schemes of enzyme stabilization and purification. We find that the details of the bacterial cultivation and isolation methods significantly affect the quality of the membranes and the protein isolated from them. Methods of bacterial cultivation and pMMO isolation were optimized such that active and purified preparations of the enzyme could be recovered. Active membrane fractions, highly enriched in pMMO and exhibiting exceptionally stable activity, were subsequently isolated using various procedures, assayed for activity, solubilized with detergents, and fractionated using available methods of protein purification. For such preparations, activity can be maintained in the membrane-bound forms for an extended period of time, a minimum of 3–4 days and up to 10 days at 4 °C with stable or enhanced activity (stable with respect to repeated freeze-thaw cycles and prolonged storage at −80 °C). Aside from describing these procedures in this report, we will discuss several other critical issues relating to the nature of the pMMO, particularly whether or not the pMMO is a copper-containing enzyme only, the subunit composition of the enzyme, and whether or not there is more than one form of the protein as suggested by recent genetic data. M. capsulatus (Bath) used in the studies were maintained on Petri plates containing the nitrate mineral salts medium with added CuSO4 (20 μm) and solidified with 1.7% agar. Cultures were maintained under an atmosphere of 20% methane in air and streaked onto fresh plates every 4–6 weeks (47Whittenbury R. Dalton H. Starr M.P. Truper H.G. Balows A. Schlegel H.G. The Prokaryotes. Springer-Verlag New York, Inc., New York1981: 894-902Crossref Google Scholar). Chemostat cultures (9–10 liters) were grown according to the following procedure. The organisms were first transferred from Petri plates to 250-ml flasks and subsequently to 2-liter Erlenmeyer flasks, containing 40 and 300 ml, respectively, of the nitrate mineral salts medium with added CuSO4 (10 μm), a 20% methane in air atmosphere, and continual shaking. The organisms were allowed to grow for 48 h in these small scale cultures. The 300-ml cultures were used to seed a fermentor containing 9 liters of the above described medium with added 20 μm CuSO4 and 20 μm CuEDTA. The methane feeding rate was controlled such that methane is growth-limiting (feeding rate ∼0.01–0.012 feet3/h·liter). The methane/air ratio was 1:4. A cell density of >10 g/liter of culture can be obtained at a higher methane feeding rate. However, the employed methane feeding rate, termed as methane stress condition (semistarvation growth condition), results in less biomass; typically only ∼5–6 g of wet cells/liter would be obtained. However, this condition was found to stimulate the overproduction of the intracytoplasmic membranes, which also contain exceptionally high levels of the pMMO (see below). Furthermore, it also stimulates copper uptake (high copper/protein ratio), resulting in exceptionally high pMMO specific activity. Approximately 24 h after inoculation and 6 h prior to cell harvest, additional CuSO4 (or CuEDTA) was added to bring the total added copper concentration to 50 and 60 μm, respectively. Six h and 3 h prior to cell harvest, the methane feeding rate was increased incrementally to 0.03–0.04 feet3/h·liter to relieve the starvation condition (partly to increase cell density). Without this step, the activity was not stable although the membranes contained unusually high levels of the pMMO. M. capsulatus (Bath) was grown at 42 °C. The pH must be maintained at 6.8–7.4 during growth. Cells were harvested in late log phase (typically 48–52 h after inoculation) by centrifugation at 27,000 × g for 15 min and washed twice with 50 mm Pipes (pH 7.2). Washed cells were suspended in lysis buffer containing 50 mmPipes, 4 mm ascorbate, 50 μg of catalase/ml of buffer, pH ∼7.2 (typically 60 g of wet cells and buffer to a volume of ∼75 ml of cell suspension). Cu(II) ions (100 μmCuSO4) can also be added to this buffer to improve the enzyme stability further. However, the addition of copper often caused the ascorbate-containing buffer to lose its effectiveness rather quickly and would complicate metal content analysis of the purified protein, so it was routinely omitted. Cell suspensions (∼0.8–1.0 g of cells/ml) were passed three times through a French pressure cell at 20,000 p.s.i. to separate the cytosolic and membrane fractions. Less dense cell suspensions (<0.5 g of cells/ml) often result in low activity or completely inactive cell-free extract and membranes. It appeared that the dense cell suspensions used here (i) kept the dioxygen tension low, hence minimizing copper oxidation and (ii) resulted in highly viscous lysate, which helped to protect the integrity of the membrane-bound pMMO during the isolation process. Unlysed cells and cell debris were removed by centrifugation at 27,000 × g for 40 min. The supernatant was then ultracentrifuged at 220,000 × gfor 90 min to pellet the membrane fraction. The clear supernatant obtained after ultracentrifugation was used as the cytosolic fraction. The pelleted membranes often show distinct layers. The minor bottom layer containing bluish and black materials and the thin, white top layer were discarded. Only the middle layer, or the translucent intracytoplasmic membranes, constituting the bulk of the membrane fractions, were collected. These membranes can be separated further on the basis of their texture into "soft" and "hard" membranes, albeit with difficulty. The difference between these two types of membranes is not great although the hard membranes appear to have higher intact pMMO content. The translucent membranes were washed by suspending them in washing buffer containing 50 mm Pipes, 5 mm ascorbate, 25 μg of catalase/ml (pH 7.2) using a Dounce homogenizer, repelleted by ultracentrifugation, and resuspended in washing buffer of 2–3 times the volume of the original cell suspension. This process was repeated a few more times until the supernatant was virtually free of soluble proteins. Finally, the pelleted membranes were suspended in storage buffer (low ionic strength storage buffer, 20–25 mm Pipes, 5 mmascorbate, 25 μg of catalase/ml of buffer, pH ∼7.25; or high ionic strength storage buffer, 75–100 mm Pipes, 50 mm imidazole, 5 mm ascorbate, 25 μg of catalase/ml of buffer, pH ∼7.25) in a volume equal to the original cell suspension volume. Sucrose (200 mm) can also be added to the above buffers to improve stability further. The membrane suspensions then can be kept at 4 °C or frozen at liquid nitrogen temperature and stored at −80 °C for future use. It should be noted that activ
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