From the Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, California 91125
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ABSTRACT |
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The particulate methane monooxygenase
(pMMO) is known to be very difficult to study mainly due to its unusual
activity instability in vitro. By cultivating
Methylococcus 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 -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,
,
, and
, 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,
',
, and
, with an apparent
molecular mass of 35, 26, and 23 kDa, respectively. The
and
'
subunits from these two forms of the pMMO contain identical N-terminal
sequences. The
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.
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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.
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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.
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 × g for 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 mM ascorbate, 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 atMembrane 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 (Mr
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.
Membrane-bound quinones were extracted using an ethanol/n-hexane mixture (2:5, v/v). The extracts were combined, dried over MgSO4, and decanted. After solvent removal, the isolated quinones were reduced by sodium borohydride. The resulting quinols, obtained as precipitates, were washed with a minimum amount of water and then with ethanol, 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 (Mr 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.
Each of the reductants was added to the membrane suspensions to give a final concentration of 5 mM in a total volume of ~1.0 ml. The assay was performed at 45 °C, and at ~5-7-min intervals, a 1-µl aliquot of the solution was removed and injected directly onto a gas chromatograph for chemical analysis. Oxidation products were identified and quantified by GC using a flame ionization detector. The activity of the pMMO was determined from the limiting initial slope of product(s) versus time plot. Specific activity was then obtained by dividing the activity by the total amount of protein in the sample as determined by the Lowry method.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.
The purified protein were first subjected to SDS-PAGE (12.5%) using the protocols described above. The proteins were then blotted into Immobilon-P membranes using the TransBlot apparatus (Bio-Rad) with a modified procedure in which the SDS concentration in the Tris/glycine transfer buffer was at least 0.2% (49, 50). Upon staining the Immobilon-P membranes with Coomassie Blue to visualize the polypeptides, the bands corresponding to each subunit were excised, and the N terminus sequence was determined using the Edman degradation method.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.
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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).
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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.
In light of these considerations, we have elected to develop either an anaerobic procedure or an aerobic procedure that can be carried out under an environment where copper oxidation is minimized during solubilization and isolation. Accordingly, in our protein isolation and purification experiments, copper oxidation was minimized by using deoxygenated buffer, performing manipulations in an anaerobic chamber, and by adding dithionite (2-5 mM) to the buffers to remove dissolved oxygen. Indeed, our first successful attempt at recovering activity from purified pMMO was achieved using a protocol in which degassed buffer was used with an excess of ascorbate to reduce all the copper ions in the preparation. Another unusual feature of the pMMO system is that the enzyme is overexpressed in the membranes. As such, the main purpose of the isolation procedure is not to enrich the pMMO several hundred- or thousand-fold, as is commonly done for many other proteins and enzymes, but the objective is to remove other contaminating proteins in the membranes, particularly heme proteins and other iron-containing proteins, as much as possible. Once purified, we relied on reconstitution experiments on these pMMO preparations to optimize the recovered activity.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.
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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).
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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.
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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.
This proton release phenomenon is linked to dioxygen tension (hence to copper oxidation). In several preparations, when treating these membranes with pure dioxygen (even prolonged storage at 4 °C, thus allowing more reaction time), a drop in pH is observed, and all pMMO activity is lost. Adding reductants (ascorbate or NADH) or deoxygenating the membranes does extend the life of these preparations, suggesting a link between dioxygen tension and copper oxidation with the observed proton release and activity loss. At the moment, it appears that the reaction of dioxygen with pMMO in the absence of substrates triggers the release of internal protons in this form of the pMMO. The source of these protons is yet to be determined, although they may be associated with glutamate/aspartate side chains that are not ligated to copper ion(s), as would be the case when the protein is not loaded with its full complement of copper cofactors. On the basis of the buffer strength (20 mM Pipes, pKa ~6.8), the magnitude of the pH drop (from 7.2 to 6.6), and the protein concentration (~50 mg/ml), the level of proton release is estimated to be ~10 H+/94-kDa protein monomer. On the basis of results obtained to date, three kinetically distinct pMMO forms can be distinguished. Type I pMMO is the stable form. This form exhibits moderate but stable specific activity in vitro and is also stable with respect to repeated freeze-thaw cycles and prolonged storage at ![]() |
ACKNOWLEDGEMENTS |
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We thank Prof. Mary E. Lidstrom and Drs. Andrei Chistoserdov, Ludmila Chistoserdova, and Roopa Ramamorthi for helpful discussions and Dr. Peter Green for assistance with the inductively coupled plasma-mass spectroscopy analysis.
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FOOTNOTES |
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* This work was supported by NIGMS, National Institutes of Health, Grant GM 22432 (to S. I. C.). Unrestricted financial support was also received from the George Grant Hoag Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a W. R. Grace fellowship and a National Research
Service Predoctoral Award.
§ To whom correspondence should be addressed: Noyes Laboratory of Chemical Physics 127-72, California Institute of Technology, Pasadena, CA 91125. Tel.: 626-395-6508; Fax: 626-578-0471; E-mail: chans{at}cco.caltech.edu.
1 The 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.
2 S. J. Elliott and S. I. Chan, unpublished data.
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