1 Department of Biochemistry, Biophysics and Molecular Biology, 4164 Molecular Biology Building, Iowa State University, Ames, IA 50011-3211, USA
2 Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226, USA
3 Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, MI 48109-2125, USA
4 Department of Chemistry, University of Wisconsin-Eau Claire, Eau Claire, WI 54702, USA
Correspondence
Alan A. DiSpirito
aland{at}iastate.edu
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ABSTRACT |
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Supplementary material is available with the online version of this paper.
Present address: Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, NE 68588-0664, USA.
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INTRODUCTION |
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Previous studies from this laboratory have shown that high-activity pMMO preparations require co-purification of Cumb with the three pMMO polypeptides (Choi et al., 2003). Additional evidence for a potential involvement of Cumb in methane oxidation comes from the culture conditions used to stabilize cell-free pMMO activity, which result in an increased concentration of membrane-associated Cumb (Choi et al., 2003
; Zahn & DiSpirito, 1996
). Studies on the role of Cumb in methane oxidation by the pMMO have been limited, as the only direct correlation between these two proteins was the irreversible loss of methane-oxidation activity following dissociation. Cumb has been shown to have superoxide dismutase activity, which may account for its stabilizing effects on cell-free pMMO activity (Choi et al., 2003
). Recent improvements in the stabilization of the pMMO in cell-free fractions (Basu et al., 2003
; Choi et al., 2003
; D. W. Choi, Y. S. Young, J. D. Semrau, W. E. Antholine, C. J. Kisting, S. C. Hartsel & A. A. DiSpirito, unpublished results), as well as in the isolation of mb (this report) and Cumb (Kim et al., 2005
), however, have caused us to reconsider the potential role of mb and Cumb in methane oxidation. In this study, we show that Cumb stimulates pMMO activity, and the results suggest that the stimulation is due to an increased rate of electron flow to the type II Cu(II) centre(s) of the pMMO.
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METHODS |
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Isolation of mb.
Cumb and mb were prepared from the spent medium of Methylosinus trichosporium OB3bT or Methylococcus capsulatus Bath. For each harvest, the spent medium was centrifuged twice at 9000 g for 20 min to remove residual cells. At this stage, the spent medium was either loaded onto a 7x20 cm Dianion HP-20 column (Supelco) or stabilized by the addition of copper as described by Kim et al. (2004), except that the final concentration of added copper was reduced from 10 to 1 mM. The Dianion HP-20 column was washed with two column volumes of H2O, eluted with 60 % methanol : 40 % water (v/v) and lyophilized. At this stage of purification, mb represented >97 % of the material absorbing at 214 or 280 nm and no other chromophores with absorption maxima above 280 nm were present. Purity of mb samples was checked at this stage by HPLC, matrix-assisted laser desorption ionizationtime-of-flight (MALDI-TOF) mass spectrometry and UVvisible absorption spectra. Unless indicated, the freeze-dried samples following chromatography on Dianion HP-20 columns were the source of mb or Cumb used in this study. Selected samples were purified further by reverse-phase HPLC on a Beckman Gold HPLC system by using a SupelcoSil LC-18 (25 cmx4·6 mm, 5 µm) column at a flow rate of 1·0 ml min1, with 10 mM sodium phosphate buffer, pH 6·6 (solvent A) and 80 % acetonitrile/H2O (solvent B) as the mobile phase. A linear gradient consisting of an initial solvent B concentration of 5 % following injection to 35 % solvent B at 50 min and 100 % at 55 min was used in this purification step.
Sample purity and metal content of final samples were based on the UVvisible absorption spectra, on metal analysis and on molecular masses as determined by MALDI-TOF MS, of the fractions before and after separation by reverse-phase HPLC.
Molecular-mass determinations.
Solution molecular mass of mb samples was determined on a Superdex Peptide HR 10/30 column (Pharmacia/LKB) equilibrated with MilliQ water (Millipore), pH 6·8. The column was calibrated by using blue dextran, orange G, bradykinin (1240 Da), rennin substrate (1759 Da), insulin (5734 Da) and horse-heart cytochrome c (12 500 Da).
MALDI-TOF mass spectra were obtained on a Voyager-DE PRO Biospectrometry Workstation 6075 (PerSeptive Biosystems, Inc.). Analyses were performed in the reflector-positive mode with time-delayed extraction (200 ns). Acquisition-mass range was typically between 500 and 5000 Da, with laser intensities between 1900 and 2100 intensity units. The matrix solution used was p-nitroaniline (Fluka Chemika) (35 mM in a 1 : 1 mixture of water : ethanol at pH 6·5). Typically, 1 µl of an mb solution (5 mg ml1) was diluted 1 : 10 with p-nitroaniline matrix. A 2 µl aliquot of the analyte/matrix solution was spotted onto a stainless-steel sample plate and allowed to dry before analysis.
Enzyme activity, isolation of cell fraction and protein determinations.
Methane monooxygenase (MMO) activity was determined by the epoxidation of propylene, as described previously (Choi et al., 2003), and measured either in the liquid phase on an SRI 8610C GC system (SRI Instruments) equipped with a flame-ionization detector and an 8'x0·085'' HaySep D column, or in the gas phase on a Varian 3900 (Varian Corporation) equipped with a flame-ionization detector and a 30 mx0·53 mm Supel-Q plot column. Isolation of the cell-free fraction, copper determinations and protein determinations were carried out as described previously (Choi et al., 2003
). In addition to propylene-oxidation activity in the soluble fraction, soluble MMO (sMMO) activity was monitored by the formation of naphthol from naphthalene as described by Brusseau et al. (1990)
.
The effects of mb on pMMO activity were examined in the washed membrane fraction from Methylococcus capsulatus Bath by using mb from Methylosinus trichosporium OB3bT. Washed membrane fractions from Methylococcus capsulatus Bath were used, as procedures for the isolation of membrane fractions with high pMMO activity have only been developed in this species (Basu et al., 2003; Choi et al., 2003
; Yu et al., 2003
). mb from Methylosinus trichosporium OB3bT was used in these studies for a variety of reasons. First, the EPR spectra of Cumb from both methanotrophs were identical (W. E. Antholine, D. W. Choi, Y. S. Young & A. A. DiSpirito, unpublished results). Second, stimulation of pMMO activity by Cumb was 1020 % higher using Cumb from Methylosinus trichosporium OB3bT than that observed with equimolar concentrations of Cumb from Methylococcus capsulatus Bath (results not shown). Third, the yields of mb from the spent medium of Methylosinus trichosporium OB3bT were generally several-fold higher than observed with Methylococcus capsulatus Bath (Choi et al., 2003
; DiSpirito et al., 1998
; Kim et al., 2005
; Zahn & DiSpirito, 1996
). Fourth, the mb from Methylosinus trichosporium OB3bT is the best-characterized mb, structurally (Kim et al., 2004
, 2005
), spectrally (D. W. Choi, Y. S. Young, J. D. Semrau, W. E. Antholine, C. J. Kisting, S. C. Hartsel & A. A. DiSpirito, unpublished results; DiSpirito et al., 1998
; Kim et al., 2005
) and thermodynamically (D. W. Choi, Y. S. Young, J. D. Semrau, W. E. Antholine, C. J. Kisting, S. C. Hartsel & A. A. DiSpirito, unpublished results). Lastly, the Cumb from Methylosinus trichosporium OB3bT is more soluble than Cumb samples from Methylococcus capsulatus Bath, which tend to precipitate in solutions at concentrations above 10 µM.
Cumb and substrate effects on the EPR spectra of washed membranes.
Membrane samples isolated under anaerobic conditions from Methylococcus capsulatus Bath were prepared for EPR studies in 6 ml amber serum vials in an anaerobic chamber (Coy Laboratory), using 5 % hydrogen with 95 % argon. In samples containing reductants (either NADH or duroquinol), 2·12 nmol reductant was added (mg membrane protein)1. The concentration of reductant added was based on an estimated pMMO content of 20 % in the washed membrane samples. For samples containing added Cu(II) or Cumb, the optimal molar ratio of Cu(II) or Cumb to pMMO was chosen based on the concentration yielding the highest propylene-oxidation activity. The samples were mixed and the hypo-vials were sealed with Teflon-coated silicon septa. Substrates (2 ml O2, 2 ml CH4 or 2 ml of each) were added with gas-tight syringes and the samples were incubated for 5 min at room temperature with shaking. After incubation, samples were transferred to EPR tubes, sealed and taken out of the anaerobic chamber. Samples in EPR tubes were then frozen in liquid nitrogen and stored on dry ice.
UVvisible absorption spectroscopy.
UVvisible absorption spectroscopy was performed as described previously (Choi et al., 2003). Kinetic photodiode array spectral series were taken by using a microvolume stopped-flow reaction analyser (Applied Photophysics and SX.18MV). Spectral series were measured at 2·0 °C from 275 to 500 nm by using a diode array detector with an integration time of 2·56 ms. The mixing chamber had a 1·0 cm path length and the monochrometer slit width was fixed at 1·0 mm entry and 1·0 mm exit. All samples were protected from ambient light to prevent possible photo-oxidation. Pro-K SVD and global analysis software from Applied Photophysics was used for data analysis (Henry & Hofrichter, 1992
).
EPR spectrospcopy.
Q-, S- and X-band EPR spectra were obtained as described by Yuan et al. (1999).
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RESULTS |
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Effects of Cumb on pMMO activity
The effects of Cu(II), mb, Cumb and mb plus Cu(II) at different molar ratios on pMMO activity in Methylococcus capsulatus Bath are shown in Fig. 1. The cells and membrane fractions used in this component of the study were from Methylococcus capsulatus Bath cultured in medium with a final copper concentration of either 60 µM CuSO4, where the expression levels of pMMO are highest, or 80 µM CuSO4, which has been shown to saturate the cells with copper (Choi et al., 2003
). The copper and mb concentrations in membrane samples of cells cultured under these conditions were approximately 250 nmol Cu (mg protein)1 (Choi et al., 2003
) and 150 nmol mb (mg protein)1. Even in cells cultured under these high-copper conditions, the addition of Cu(II) usually stimulated pMMO activity, although higher concentrations of Cu(II) were always inhibitory (Fig. 1a
). In whole-cell samples, the stimulation of pMMO by copper was three- to fourfold higher if added as Cumb (Fig. 1a
). An optimal Cumb-to-cell ratio was always observed, followed by a small decrease in pMMO activity as the Cumb-to-cell ratio was increased further. However, even at high Cumb concentrations, the addition of Cumb stimulated pMMO activity in whole-cell samples and never showed the inhibition observed with Cu(II). This stimulation was only observed with Cumb; mb itself was slightly inhibitory to whole-cell propylene-oxidation rates (Fig. 1a
).
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Several recent experiments have suggested that mb may initially bind copper as a homodimer, i.e. as Cu(mb)2, followed by the binding of a second Cu(II), resulting in a final molar ratio of 1 copper atom per mb, i.e. Cumb (D. W. Choi, Y. S. Young, J. D. Semrau, W. E. Antholine, C. J. Kisting, S. C. Hartsel & A. A. DiSpirito, unpublished results). To examine whether the pMMO in the washed membrane fraction responded differently to Cu(mb)2, the effect of Cu(mb)2 on pMMO activity was also examined (Fig. 1c). In contrast to Cumb, which stimulated pMMO activity, Cu(mb)2 was inhibitory to pMMO activity (Fig. 1c
). To further examine this property, the effect of mb with different copper-to-mb molar ratios on pMMO activity in the washed membrane samples was examined (Fig. 1d
). In general, mb was inhibitory to pMMO activity at copper-to-mb molar ratios of <0·6 copper atoms per mb and stimulatory at concentrations above 0·60·8 copper atoms per mb (Fig. 1d
).
EPR spectra of mb
In an attempt to identify Cumb spectrally in the washed membrane fraction of methanotrophs, the EPR spectral properties of purified mb were examined by X-, Q- and S-band EPR. The EPR spectrum of Cumb at the conventional microwave frequency (X-band) and at a higher (Q-band) and a lower (S-band) frequency confirms the binding of cupric ion (Fig. 2). Two observations distinguish the X-band spectrum of Cumb. First, the lines in the low-field region [gll=2·23, All=185 G (1 G=104 T)] were broader than usual (Boas, 1984
). This indicates more strain in the axial direction than is observed from most type II cupric complexes. These lines in the gll region were sharper at a lower microwave frequency (S-band trace in Fig. 2
) and broader at higher microwave frequencies (Q-band). For Q-band analysis, the gll lines were broad and not detected (not shown). In the X-band spectrum, there were lines at high field, split by 16 G. These lines split by 16 G were also evident in the S-band spectrum on the S-shaped signal from the gl region. The first harmonic of the S-band trace emphasizes the sharp lines. The Q-band spectrum also has sharp lines on the high-field side, which are attributed to the g
region. Superimposed on the Cumb lines were five or six Mn lines and a free-radical signal that were not detected at X- or S-band. Q-band spectra contained Mn and free-radical signals that were considered background signals. The first harmonic of the Q-band spectrum emphasizes the sharp lines. They are part of the g
region from an axial-symmetric site and not from gx for a rhombic site with three g values (gz, gy, gx), because the gx peak would be superimposed about the free radical in the Q-band spectrum if this was a true g value. Presumably, the shoulder on the high-field side in the X-band spectrum was an overshoot line that disappears in the Q-band spectrum, as expected for an overshoot line at X-band. As there were at least 10 lines split by 16 G that were resolved and probably more unresolved lines in the spectra, these lines were attributed to superhyperfine lines due to nitrogen-donor atoms in addition to protons that are close to the cupric ion. It is surmised that the cupric-binding site was formed from three or four nitrogen-donor atoms, due to the number of lines resolved, the gll value of 2·23 and the All value of 185 G.
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Cu(II), Cumb and substrate effects on the EPR spectra in membrane samples
The stimulatory effect of Cumb on propylene oxidation by the pMMO suggests a potential role of Cumb in methane oxidation by the pMMO. To examine the possible cause for this stimulation, the EPR spectral properties in the washed membrane fraction were examined following the addition of Cumb in the presence and absence of a reductant, NADH, and substrates, CH4 and/or O2. To minimize variability in the spectral characterization of the pMMO, the membranes were isolated and reaction vials were prepared under anaerobic conditions. In reaction mixtures containing O2 and/or CH4, the gases were added with gas-tight syringes to closed 6 ml serum vials in the anaerobic chamber. Under these conditions, the EPR-detectable Cu(II) in the pMMO was reduced by 5065 % and the addition of NADH did not result in additional reduction of the type II Cu(II) centres associated with the pMMO [Fig. 5a, traces (i) and (ii)]. The inability to reduce the remaining copper centres may represent the physiological resting state of the enzyme or it may represent the population of inactive enzyme in these preparations. Addition of CH4 did not change the spectral properties [Fig. 5a
, trace (iii)]. However, in the presence of O2, the intensity of the type II Cu(II) signal was increased by approximately 50 % and a free-radical signal was generated at g=2·005 [Fig. 5a
, trace (iv)]. The addition of methane and oxygen resulted in spectra that were similar to the spectra of O2 alone, but the free-radical signal was reduced by 40 % [Fig. 5a
, trace (v)]. These results suggest that, as in the sMMO (Wallar & Lipscomb, 1996
, 2001
), the pMMO activates oxygen before reacting with methane.
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In other membrane preparations, the reduction of the pMMO following addition of Cumb was comparatively small (Fig. 5c). Also, as in the previous example (Fig. 5b
), the addition of high concentrations of Cumb did not increase the intensity of the Cu(II) signal in the washed membrane sample, suggesting that the copper associated with mb remained as Cu(I). In these membrane preparations, the intensity of the radical signal became larger following O2 addition [Fig. 5c
, trace (iv)]. However, in contrast to the membrane series represented in Fig. 5(b)
, the addition of CH4 with O2 only resulted in partial quenching of the radical signal [Fig. 5c
, trace (v)]. As a general rule, Cu(II) is known to stimulate pMMO activity in whole-cell as well as in cell-free fractions (Basu et al., 2003
; Chan et al., 2004
; Choi et al., 2003
; Collins et al., 1991
; Dalton et al., 1984
; Lieberman et al., 2003
; Nguyen et al., 1998
; Zahn & DiSpirito, 1996
). However, in contrast to the copper added as Cumb, much of the added Cu(II) appeared as unassociated copper, which becomes more evident following the addition of O2 or O2 plus CH4 (results not shown).
Effects of mb on the cupric signal in the g region
One surprising result in the examination of membrane samples was the absence of the normally well-resolved superhyperfine structure in the g region associated with the type II Cu(II) of the pMMO (Nguyen et al., 1996
; Yuan et al., 1997
, 1998a
, b
, 1999
) [Fig. 5b
, traces (i) and (v)]. This phenomenon was only observed in membrane preparations from cells cultured in high-copper, i.e. 80 µM CuSO4, medium, which has been shown to copper-saturate Methylococcus capsulatus Bath (Choi et al., 2003
; Zahn & DiSpirito, 1996
). To examine whether Cumb was responsible for the loss of superhyperfine structure associated with the pMMO, the effect of mb at different copper-to-mb ratios on the EPR spectra from washed membrane samples was examined. Fig. 6
(a) shows the EPR spectra of the washed membrane sample from cells cultured in 80 µM CuSO4 medium. In these membrane samples, the type II Cu(II) superhyperfine structure was initially well-resolved (Fig. 6a
, no-addition trace). However, the addition of mb containing copper-to-mb ratios above 0·25 Cu per mb resulted in the loss of copper superhyperfine structure, as well as the free-radical signal, at g=2·005. At copper-to-mb ratios above 0·25 Cu per mb, signal intensity increased in the cupric spectral region and was similar to Cu(II)-titration experiments with purified mb. Fig. 6(b)
shows the EPR spectra of washed membrane samples from cells cultured in 60 µM CuSO4, where maximal expression of the pMMO has been observed (Choi et al., 2003
; Zahn & DiSpirito, 1996
). In these membrane samples, the addition of mb with higher Cu(II)-to-mb ratios decreased, but did not eliminate, the resolution of the superhyperfine structure in the g
region. The results suggest that the loss of cupric superhyperfine structure in some membrane preparations was the result of the high Cumb concentrations. We speculate that the decrease in the resolution of the superhyperfine structure resulted from reduction and/or presence of multiple signals in this region. Increased resolution of the superhyperfine signal following the removal of Cumb has also been observed in purified pMMO preparations (Choi et al., 2003
).
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DISCUSSION |
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The EPR and kinetic experiments described here suggest that Cumb is a redox-active chromopeptide that stimulates methane oxidation by pMMO. This stimulation was equal to or greater than that observed with Cu(II) and without the toxicity observed at higher Cu(II) concentrations. The stimulation of pMMO activity was similar to the stimulation observed previously with P-centre inhibitors of the bc1 complex, such as mixothiazol and stigmatellin (Brand & Trumpower, 1994; Choi et al., 2003
; DiSpirito et al., 2004
; Matsumo-Yagi & Hatefi, 1999
; Zahn & DiSpirito, 1996
; Zhang et al., 1998
). Stimulation of pMMO activity by mixothiazol and stigmatellin has been interpreted as resulting from either the preferential shuttling of electrons to the pMMO or that the pMMO has a quinone- or semiquinone-binding site (DiSpirito et al., 2004
). Most of the available evidence supports the second interpretation (Basu et al., 2003
; Choi et al., 2003
; DiSpirito et al., 2004
; Shiemke et al., 1995
, 2004
; Zahn & DiSpirito, 1996
).
The EPR studies presented here suggest interactions between Cumb and the type II Cu(II) centre of the pMMO and Cumb. The results also suggest that Cumb is the probable source of the variability, complexity and controversy associated with the copper centres of the pMMO (Basu et al., 2003; Chan et al., 2004
; Choi et al., 2003
; Lieberman et al., 2003
; Lieberman & Rosenzweig, 2004
, 2005
; Nguyen et al., 1994
, 1996
, 1998
; Takeguchi & Okura, 2000
; Takeguchi et al., 1999
; Téllez et al., 1998
; Yuan et al., 1997
, 1998a
, b
, 1999
; Zahn & DiSpirito, 1996
). Laboratories examining the pMMO have differed in the analysis of the EPR spectra. Results from studies using the washed membrane fraction from Methylomicrobium album BG8 (Yuan et al., 1997
, 1998a
, b
, 1999
) have suggested that the main, if not sole, source of the EPR spectrum is from a type II Cu(II) site. Other laboratories examining pMMO in Methylococcus capsulatus Bath have suggested that the spectrum associated with the pMMO is the sum of two EPR signals, one from a type II Cu(II) site and a second either associated with Cumb (Choi et al., 2003
; Zahn & DiSpirito, 1996
) or from a trinuclear Cu(II) cluster (Chan et al., 2004
; Nguyen et al., 1994
, 1996
, 1998
). mb from Methylomicrobium album BG8 has also been isolated recently (D. W. Choi, Y. S. Young & A. A. DiSpirito, unpublished results). However, the concentrations of Cumb in the washed membrane fractions from Methylomicrobium album BG8, cultured in NMS medium containing 510 µM CuSO4, were <10 % of the concentration observed in the membrane fractions from either Methylococcus capsulatus Bath or Methylosinus trichosporium OB3bT. The lower concentration of Cumb in Methylomicrobium album BG8 may account for the less-complex and better-resolved superhyperfine structure in the g
region in samples, as previous studies have shown an increased resolution of superhyperfine structure in the g
region following separation of Cumb from the
subunits of the pMMO from this organism (Choi et al., 2003
). Taken together, the results support the view that the site of the second EPR signal is from Cumb.
In conclusion, the results presented here provide the first direct evidence for the role of Cumb in methane oxidation by the pMMO. The exact mechanism of stimulation is still unknown, but the results suggest that Cumb increases electron flow to the type II Cu(II) centre(s) in the pMMO and may be involved in radical formation. Cumb may also have a secondary role in protection against oxygen radicals and/or delivery of copper to the pMMO, as speculated earlier (Choi et al., 2003; DiSpirito et al., 2004
).
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ACKNOWLEDGEMENTS |
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Received 29 April 2005;
revised 15 July 2005;
accepted 20 July 2005.
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