INTRODUCTION

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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 (1, 2). 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 (3). Two distinct species of methane monooxygenase (MMO)¹ are known to exist at different cellular locations, a cytoplasmic (soluble) MMO and a membrane-bound (particulate) MMO (4). The soluble MMO (sMMO) is a complex three-component system consisting of a hydroxylase, a reductase, and a small regulatory protein (4). The sMMO has been investigated extensively by several research groups (5-21). The x-ray crystal structure of the sMMO hydroxylase isolated from Methylococcus capsulatus (Bath) has been solved (22, 23). The hydroxylase active site contains a non-heme binuclear iron cluster. In contrast, the particulate methane monooxygenase (pMMO) appears to be a copper protein (24-29). 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 (1, 2, 4). 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 (6, 30-34). In strains capable of expressing either the sMMO or pMMO, the sMMO is expressed under copper stress only (low copper/biomass ratio) (35-39). 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 (35-39). 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 (35-39).

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 (24-26, 28, 29, 40). Activity was found to be proportional to the level of membrane-bound copper ions (24, 27-29). The pMMO-associated copper ions appear to be organized into trinuclear cluster units with rather defined magnetic and redox properties (24-26, 28, 29, 40). 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 (25, 26, 28, 29, 40). 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 (41, 42). 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 (24-26, 28, 29, 40). 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 (43). 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 (44). According to the authors, any attempts to purify the enzyme further resulted in complete loss of activity. A few reports including a recent work (45, 46) 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 from M. 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.

	MATERIALS AND METHODS

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Growth of Methanotrophs and Membrane Isolation-- 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 (47). 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 mM Pipes, 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 µM CuSO₄) 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.

Membrane Solubilization-- The membrane suspension was first degassed by several vacuum/argon cycles. The membranes in storage buffer (in either low or high ionic strength buffer) were then treated with either solid or 20% (w/v) stock solution of dodecyl -D-maltoside (to a final concentration of 3-5% (w/v) or ~2 mg of detergent/mg of protein). The mixture was mixed rigorously and incubated on ice for 30 min to 1 h and then centrifuged at 37,000 × g for 45 min to remove unsolubilized materials. The clear supernatant was taken as the solubilized membranes, and used for subsequent steps.

Rapid Isolation Procedure Using L-Lysine-Agarose Affinity Chromatography and Removal of Positively Charged and Iron-containing Proteins-- The L-lysine-agarose column (Sigma) (20 × 2 cm) was equilibrated with buffer containing 25 mM Pipes, 5 mM ascorbate, with or without 200 µM CuSO₄ and 0.05% (w/v) dodecyl -D-maltoside, pH ~7.25. Dithionite (5 mM) can be used in lieu of ascorbate; however, strict anaerobic protocol must be followed. The solubilized membranes (~2 ml; ~40-60 mg of total protein) were applied to the column, and 0.5-ml effluent fractions were collected, employing an elution buffer of 20-25 mM Pipes, 5 mM ascorbate, 0.05% (w/v) dodecyl -D-maltoside, pH 7.2, but with no sucrose or imidazole added. The elution rate from the column was typically 0.5-1.0 ml/min. Three to four fractions can be obtained. The flow-through fraction contains large pieces of the solubilized membranes and most of the positively charged proteins. A fast moving fraction contains several proteins (heme-containing proteins) but also pMMO (purity of ~70% or higher). Next, a slow moving fraction constitutes the bulk of the solubilized membranes and contains mostly the three-subunit form of the pMMO (purity of ~90% or higher). Finally, a minor binding fraction can be eluted out of the column using buffer containing 50 mM Pipes, 100 mM NaCl, pH ~7.25. This binding fraction consists of mostly heme-containing proteins but also some residual pMMO.

Large Scale Isolation Procedure Using Anion Exchange Chromatography and Removal of Positively Charged and Iron-containing Proteins-- Large scale isolation of the enzyme can also be obtained with a variation of the above procedure using DEAE-Sepharose Fast Flow (Amersham Pharmacia Biotech). A DEAE-Sepharose Fast Flow column was equilibrated with buffer containing 100 mM Pipes, 50 mM imidazole, 5 mM ascorbate, 200 µM CuSO₄, 0.05% (w/v) dodecyl -D-maltoside buffer at pH ~7.25. Sucrose (200 mM) can also be included in this buffer, but its effectiveness is not great. 5-7 ml of solubilized membranes (concentration 20-30 mg/ml) in high ionic strength storage buffer were then applied to the column. When an anaerobic protocol was used, the column was first degassed, dithionite (5 mM) was added to the equilibrating buffer to remove dissolved dioxygen, and the manipulations were performed in an anaerobic chamber. The column was then washed with one column volume of the equilibrating buffer. DEAE-Sepharose FF column fractionates the solubilized membranes into four fractions. There are two flow-through fractions, a fast moving fraction (~10 ml) containing a mixture of two forms of pMMO (see below) and a slow moving fraction (~10-20 ml) containing positively charged proteins (proteins of high pI), a truncated form of pMMO, and other impurities. The bound proteins are eluted out using the above high ionic strength buffer combining with a NaCl (or NH₄Cl) gradient from 0 to 200 mM. They are separated into two fractions. The fraction eluted out at <100 mM NaCl (~200 ml) contained mostly the pMMO as judged from the SDS-PAGE assay (purity >90%). The second fraction (50 ml or less) eluted out of the column at higher salt concentration (>100 mM NaCl) with a characteristic low pI and low molecular mass contaminant (~22 kDa) as well as other minor impurities. The isolated proteins were concentrated using Amicon ultrafiltration membranes (M_r cut-off 50,000 or 100,000).

Lipids and Membrane-bound Quinone Isolation-- Membrane suspensions (protein concentration >20-30 mg/ml) were mixed with a methanol/chloroform (1:3, v/v) mixture. The wet membrane suspension/extraction solution mixture (typically 1:5 to 1:10, v/v) was shaken rigorously and decanted. The process was repeated at least three times to ensure complete extraction. The extracts were combined, dried over anhydrous MgSO₄, and decanted. After solvent removal, the crude yellowish lipids were dried and stored under vacuum for later use.

Protein Reconstitution-- A 2-3-ml volume of the buffer containing 10-20 mg/ml of the isolated lipids was sonicated for 10-15 min to disperse the lipids and mixed with 1 ml of purified protein in detergent-containing buffer (protein concentration 20-30 mg/ml). The resulting mixture can be sonicated briefly for a few seconds to assure dispersion. As soon as the detergent-containing protein solution was added, the solution became clear. The mixture was then loaded into dialysis tubing (M_r cut-off 50,000 or 100,000). The tube was dialyzed against a buffer containing 50 mM Pipes, 10 mM ascorbate, 200 µM CuSO₄, 100 mM (NH₄)₂SO₄ for 12 h with continuous stirring. The reconstituted protein was assayed immediately for activity and stored either at 4 °C or 80 °C for later use. The excess detergent can also be removed using BioBeads SM-2. A volume of ~2-3 ml of the purified protein (~30-50 mg/ml) was passed through a column (1 × 5 cm) of Bio Beads, and the eluate was concentrated using Amicon ultrafiltration membranes and mixed immediately with a sonicated lipid suspension (10-20 mg/ml) as described above (lipid/protein ratio 1:1 or 2:1, v/v). This mixture can be sonicated briefly for a few seconds to ensure dispersion. The reconstituted protein was then assayed for activity immediately. The BioBeads method did not prove to be a useful approach to prepare lipid-reconstituted protein, since the pMMO tended to precipitate out of the buffer as soon as the detergent was removed.

MMO Activity Assay-- The MMO activity of samples was measured by alkane substrate (propane, butane) oxidation or propene epoxidation assays. For membrane fractions, solubilized membranes, purified protein, and reconstituted pMMO, the reductant of choice was NADH. Dithionite (3-5 mM) was also found to be capable of supporting turnover; however, it must be used in conjunction with a strong buffer (100 mM Pipes) and must be assayed using alkane substrates. Duroquinol and membrane-originated quinols were tested as potential sources of reducing equivalents. Duroquinone obtained from Sigma and quinones isolated from the membranes were reduced by sodium borohydride and purified as described above.

SDS-PAGE, Protein Blotting, and N Terminus Sequencing-- Each pool of protein obtained during purification as well as the concentrated purified protein was analyzed using SDS-PAGE (12.5-15%) according to Laemmli (48). It is essential not to heat the protein in dissociating buffer before loading, since this step results in substantial degradation and cross-linking. The polypeptide bands were visualized by staining with Coomassie Blue.

Metal Assay-- Metal ion analysis (copper, iron, zinc, cobalt, manganese, and nickel) was performed by inductively coupled plasma-mass spectroscopy. The copper concentration of the samples was determined relative to standard solutions of Cu(NO₃)₂, ranging in concentration from 7.3 to 155 µM in 0.1 N HNO₃ (Aldrich). A solution of 0.1 N HNO₃ in distilled water was used as a copper-free control. Samples of purified pMMO were used as obtained or digested at 45 °C using ultrapure metal-free sulfuric acid obtained from Aldrich. The protein solution was diluted with ultrapure water containing 0.1 N HNO₃ to the appropriate concentration prior to analysis. The values reported are the averages of three separate determinations. The same samples were used for iron analysis, but the standards were prepared by diluting an iron atomic absorption standard purchased from Sigma with 0.1 N HNO₃.

UV-visible and EPR Spectroscopy-- EPR spectra were recorded on a Varian E-line Century X-band spectrometer. In the EPR experiments, sample temperature was maintained at 77 K with a liquid nitrogen Dewar or at 4.2-100 K with an ESR-900 Oxford Instruments (Oxford, United Kingdom) liquid helium cryostat. The EPR samples were prepared by sealing 200 µl of protein solution under an atmosphere of argon in quartz EPR tubes at a total protein concentration of ~50 mg/ml in 20 mM Pipes (pH 7.2). UV-visible spectra were taken using a Hewlett-Packard HP 3502A UV-visible spectrometer.

	RESULTS AND DISCUSSION

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The Overproduction of Highly Enriched pMMO-containing Membranes-- The cultivation of methanotrophs is often plagued by a foreign organism contamination, possibly another methylotroph, which thrives on the byproducts of methanotroph metabolism. As a result, samples of methanotroph chemostat cultures were routinely withdrawn and scrutinized under a microscope every 12 h to ensure the culture purity. Only absolutely pure cultures as ascertained by microscopy were subjected to membrane isolation and further experiments. At low methane feeding rate conditions as described, the release of methane metabolism by-products was minimized, and bacterial contamination was completely eliminated. In addition to eliminating bacterial contaminants, maintaining a low methane feeding rate also stimulates the overproduction of the intracytoplasmic membranes and the pMMO. We routinely obtain a minimum of 60% or more cellular mass in the form of these membranes using growth conditions as described above. In these membranes, the pMMO indeed constitutes the bulk of the membrane-bound proteins (Fig. 1).

View larger version (36K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE of pMMO-overexpressed membranes isolated from M. capsulatus (Bath). Lane 1, standards. Lanes 2 and 3, electrophoretic profile of pMMO-overexpressed membranes isolated from M. capsulatus (Bath) grown under methane stress conditions. Note the absence of high molecular weight polypeptides in these preparations.

Methodology for pMMO Isolation-- To develop a suitable protocol for successful isolation of the active enzyme, several unusual features of the pMMO had to be recognized (1, 2). The enzyme appears to require a lipid environment to function, since solubilization often results in >90% loss of activity (44). Previous attempts at isolating the protein indicated that once being removed from the lipid bilayer, the enzyme deactivates quickly. Our on-going characterization of the enzyme in situ indicates that the enzyme contains an exceptionally high level of Cu(I) ions, a unique feature for a monooxygenase (25, 26). As such, the loss of activity upon solubilization and during purification could be a result of overoxidation and subsequent loss of some of the more labile copper cofactors. In the protocols for isolating the sMMO hydroxylase developed by Fox et al. (5), the reduced form of iron was introduced into the isolating buffer, resulting in preparations with a 10-20-fold increase in activity. Similar approaches may not work for the pMMO, a copper-containing protein, since an air-stable Cu(I) complex with a high dissociation constant is not readily available. Despite the fact that Cu(II) ions have been found to improve the pMMO activity for certain pMMO preparations, the bulk of the copper ions associated with the pMMO actually exists in the reduced Cu(I) form. As Cu(I) ions are extremely insoluble in aqueous solution, these copper sites are relatively inert. However, once oxidized in an aerobic environment, they can become quite labile, particularly for those copper cofactors that are relatively exposed.

Components of the pMMO System-- Assuming that we can isolate intact pMMO with full metal content, it does not follow that we will observe optimal activity, since maximal activity may require the presence of other crucial components such as a pMMO reductase and possibly an activity-regulatory protein. The existence of a pMMO reductase is certain, since NADH, a reductant capable of supporting pMMO turnover in vitro is a two-electron donor, while each copper ion is a one-electron acceptor. This fact suggests the presence of a mediator. From the available literature, we can postulate possible types of reductase systems for the pMMO. A two-component pMMO system would consist of a pMMO hydroxylase and a reductase that accepts electrons from NADH and channels them directly to the hydroxylase, a scenario found to be the case for the sMMO. A three-component system might consist of a pMMO hydroxylase; a mediator component, which could be a species like cytochrome b or c or a quinone analog; and a NADH oxidoreductase, which accepts electrons from NADH and channels them to the mediator molecule. Depending on the nature of the mediator, this oxidoreductase could be a NADH/quinone oxidoreductase or a NADH/cytochrome oxidoreductase and could be either membrane-bound or membrane-soluble. Recent results of Shiemke et al. (51) suggest that the pMMO system might be a three-component quinone oxidoreductase/quinol/hydroxylase. Confirmation of this hypothesis can be readily made by assaying the enzyme in the form of membrane-bound, solubilized, or purified/reconstituted pMMO hydroxylase using the quinones isolated from the membranes, purified and reduced as described. However, a conclusive result has yet to be obtained. In any case, this scenario now seems rather unlikely, with the recent isolation and characterization of a flavin-containing NADH oxidoreductase that appears to be associated with the pMMO.²

Characterization of the Purified pMMO Hydroxylase Activity: Metal Content-- The major protein isolated from the membranes from now on will be referred to as the pMMO hydroxylase (pMMOH). The isolated pMMOH upon detergent removal and lipid reconstitution exhibits a significant level of recovered activity (Table I). Although the recovered activity has yet to be as high as the level in whole cells or in our most active membrane preparations to date, the level of observed activity is significant enough for us to draw important and critical conclusions regarding the nature of the pMMO.

Subunit Composition of the pMMO Hydroxylase and pMMO Polymorphism-- Purification of pMMOH from solubilized membranes and assaying of the polypeptide profile of the collected fractions demonstrates that three polypeptides are consistently co-purified together concomitant with recovered activity. This result suggests that at least one form of pMMO contains three subunits, since co-elution during purification is one of the criteria to determine the subunit association of an enzyme. SDS-PAGE analysis of the purified pMMOH fractions obtained from these experiments indicates an apparent molecular mass of 45, 26, and 23 kDa, for these subunits, named , , and , respectively (Fig. 2, lane 3). The assertion that the pMMOH contains a core of three different subunits is also supported by the observation that in several highly active and stable preparations, electrophoretic profiles of membranes (as well as purified pMMO) display only three major bands intensely, implying that these three subunits are all of the essential membrane-bound components needed for pMMO activity. The 35-kDa polypeptide observed previously in MMO expression switch-over experiments is absent in these preparations (35-39) (see below).

View larger version (84K): [in this window] [in a new window]   Fig. 2.   SDS-PAGE of fractions obtained from DEAE-Sepharose chromatography of solubilized membranes isolated from M. capsulatus (Bath). Lane 1, standards. Lane 2, fraction 1 (flow-through, fast moving fraction). Note the prominent presence of all of the 45-, 35-, 26-, and 23-kDa polypeptides. The purity of the pMMO (both forms of the enzyme) in this fraction is estimated at >70%. Lane 3, fraction 2 (flow-through, slow moving fraction). Note the near absence of the 45-kDa band; however, the 35-kDa band is still present (albeit with smearing) as well as the ~26- and ~23-kDa bands. The presence of lightly stained high and low molecular weight polypeptides can also be seen. Lane 4, fraction 3 (weakly binding fraction eluted at <100 mM NaCl or NH4Cl). This fraction is mostly the pMMO (purity >90%). The 26-kDa band exhibits some smearing. Lane 5, fraction 4 (strongly binding fraction eluted at >100 mM NaCl or NH4Cl). This fraction contains mostly a very low pI, 24-kDa polypeptide which has an N-terminal sequence of AAQASLERNL. Other minor impurities can also be seen.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   SDS-PAGE of two major fractions obtained from DEAE-Sepharose chromatography of unusual solubilized membranes isolated from M. capsulatus (Bath). Lane 1, low ionic strength fraction (<100 mM), where the 35-kDa polypeptide is prominent. The 35-, 26-, and 23-kDa band intensities are nearly the same, whereas the 45-kDa band is virtually absent. Lane 2, high ionic strength fraction (>100 mM), where the 45-, 26-, and 23-kDa band intensities are nearly the same, whereas the 35-kDa band is almost completely absent.

Spectroscopic Characterization of the Purified pMMO Hydroxylase-- The UV-visible and EPR spectra of various purified pMMO preparations are shown in Figs. 4, 5, 6, and 7. The purified pMMOH also appears not to contain any other common biological cofactors as suggested by its UV-visible absorption spectrum (Fig. 4). The UV-visible spectrum of the purified pMMOH exhibits only the protein absorption (~280-300 nm) and a very weak band at 410 nm that can be attributed to a very slight cytochrome contaminant(s). Further efforts to remove the cytochrome contaminants resulted in complete deactivation of the enzyme and hence were not pursued further.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   UV-visible spectrum of the purified pMMO that has the (45-, 26-, and 23-kDa) subunit composition. Note the presence of a very weak absorption band at ~410 nm due to minor heme contamination. The concentration was 2.3 µM (inset, 40.6 µM) at pH 7.2 in 25 mM Pipes buffer and 0.05% dodecyl -D-maltoside.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   The X-band EPR spectrum of the purified pMMO as isolated. The spectrum was recorded at the temperature of 8 K with microwave power of 0.1 milliwatt, modulation frequency of 100 kHz, modulation amplitude of 10 Gauss, and gain of 8 × 103. See "Materials and Methods" for protein concentration.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   The X-band EPR spectrum of the fully oxidized purified pMMO obtained by ferricyanide treatment. Excess ferricyanide was removed by repeated washing. The spectrum was recorded at 4 K with microwave power of 0.05 milliwatt, modulation frequency of 100 kHz, modulation amplitude of 10 Gauss, and gain of 4 × 103. See "Materials and Methods" for protein concentration.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   UV-visible spectrum of the fully oxidized purified pMMO that has the (45-, 26-, and 23-kDa) subunit composition. Note the appearance of a broad absorption band at ~500 nm in addition to the ~400-nm band observed previously in the purified as-isolated pMMO. The concentration was 3.7 µM (inset, 37 µM) at pH 7.2 in 25 mM Pipes buffer and 0.05% dodecyl -D-maltoside.

Factors Contributing to pMMO Activity Stability and Kinetically Distinct pMMO Forms-- Maintaining high copper concentrations during growth has a marked effect on the isolated membranes, particularly higher and longer lasting activity in vitro. Equally important factors are cell growth/life cycle, pH, and levels of copper oxidation in solution (which are linked to dioxygen tension). Cell cycle is critical, since we obtain preparations with stable activity only with mature cells grown on Petri plates for more than 7 days and less than 4 weeks. The pH strongly affects the activity of the enzyme. Upon exposure of highly active membrane preparations to acidic pH (pH <7, or even a short exposure to 6.8), the membranes quickly lose all of the activity. While readjusting the pH to physiological ranges (7.2), no activity, or at best base-line activity, can be observed. Interestingly, certain membrane preparations become acidic rather quickly upon direct exposure to atmospheric oxygen, and once this phenomenon occurs, all activity is lost quickly. It has become clear to us that this is the underlying reason for the unusual instability of the pMMO activity: in all instances where activity is lost at any stage (cell lysis, membrane isolation, membrane solubilization, or the thawing of frozen membranes), a significant drop in pH is always observed.