The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath

Abstract

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.

FIG. 1.

Effect of copper concentration in the culture medium on the rates of propylene oxidation in whole-cell (▵), washed membrane (○), and soluble (□) fractions. The rate of copper addition was 1.7 μM · h⁻¹, while the cell density was maintained at an OD₆₀₀ of between 1.8 and 2.0.

FIG. 2.

(A) Copper concentrations associated with the whole-cell fraction (□) and membrane fraction (○) of cells cultured in chemostats with increasing copper concentrations. The right-hand axis shows the copper concentration in the spent medium following cell separation (▵). (B) Percent increase in copper per cell (○) and copper per milligram of whole-cell protein (•). Chemostat conditions and cell samplings were as described in the legend to Fig. 1.

FIG. 3.

(I) SDS-PAGE of whole-cell samples from M. capsulatus Bath. Cell samples were taken before the addition of copper (0.3 μM copper) (lane A) and when the copper concentration in the chemostat reached 1 (lane B), 5 (lane C), 15 (lane D), 25 (lane E), 35 (lane F), 45 (lane G), 55 (lane H), 65 (lane I), and 75 (lane I) μM. Molecular mass standards (Invitrogen Mark 12 standards; 200, 116.3, 97.4, 66.3, 55.4, 36.3, 21.5, 14.4, 6, 3.5, and 2.5 kDa) are shown in lane K. Chemostat conditions and cell samplings were as described in the legend to Fig. 1. The cell sample in each lane was standardized to 1.3 × 10⁸ cells per lane. (II) α-NT, Coomassie-stained gel illustrating the protein remaining in the 43-kDa region of an SDS-polyacrylamide gel following blotting for 1 h. The Coomassie-stained gel following transfer is included because of the poor transfer of the α subunit of the pMMO. (III and IV) Immunoblot analysis of M. capsulatus Bath cell fractions with antibodies against the α (III) and β (IV) subunits of the pMMO and with antibodies against MDH. (V) MDH was used as a non-copper-regulated protein control. Arrows to the left indicate sMMO hydroxylase polypeptides, and those to the right indicate pMMO polypeptides.

FIG. 4.

Transcript concentration of pmoA (▪) and mmoX (□) in M. capsulatus Bath cells cultured in medium not supplemented with copper; expressing sMMO; and following the addition of 1, 5, 25, or 55 μM CuSO₄. Chemostat conditions and cell samplings were as described in the legend to Fig. 1.

FIG. 5.

Thin-section transmission electron micrograph of M. capsulatus Bath cultured as described in the legend to Fig. 1. Chemostat conditions were as described in the legend to Fig. 1. Cell samples were taken when the added copper concentration in the culture medium reached 5 (A), 20 (B), 40 (C), 60 (D), 80 (E), and 89 (F) μM. Marker bar, 200 nm.

FIG. 6.

Effect of copper concentration during growth of M. capsulatus Bath with low (A) and high (B) concentrations of fatty acids. The following fatty acids were monitored: 16:0 3-OH FAME (•), 14:0 FAME (○), 17:0 FAME (▵), 15:0 FAME (▴), 16:0 (▪), 16:1 cis 9 FAME (□), and 16:1 cis 11 (⧫). The chemostat conditions and cell samplings were as described in the legend to Fig. 1.

FIG. 7.

(Top) Effect of dodecyl β-d-maltoside on membrane-associated propylene oxidation activity in M. capsulatus Bath. Shown are the rates of propylene oxidation following incubation in dodecyl β-d-maltoside for 1 h under anaerobic conditions before (○) and the soluble fraction after (•) centrifugation at 150,000 × g for 90 min. (Bottom) SDS-PAGE of solubilized protein (2-μl volume) samples following 150,000 × g centrifugation of washed membrane samples incubated for 1 h with 0.3 (B), 0.5 (C), 0.7 (D), 1.0 (E), 1.4 (F), 1.8 (G), 2.0 (H), 3.0 (I), or 4.0 (J) mg of dodecyl β-d-maltoside per mg of membrane protein.

FIG. 8.

Effect of the dodecyl β-d-maltoside concentration used in the initial solubilization of pMMO on the propylene oxidation activity (□) and copper (○) and iron (▵) composition per αβγ subunit of purified pMMO.

FIG. 9.

X-band EPR spectra at 77 K of the cupric site in high-detergent-concentration-treated purified active pMMO (79 mU/mg of protein) containing 10 Cu and 2 iron atoms per αβγ complex (A) and following detergent treatment and containing 2 copper and 2 iron atoms per αβγ complex (B). The following experimental conditions were used: modulation frequency, 100 kHz; modulation amplitude, 5 G; time constant, 100 ms; microwave frequency, 9.191 GHz; and microwave power, 5.0 mW.

FIG. 10.

Inhibition of nitroblue tetrazolium reduction in the presence of Cu-cbc.

FIG. 11.

SDS-PAGE (slab gel) of fractions during the purification of NDH-pMMO complex and of pMMO from M. capsulatus Bath. Lanes: A, whole-cell fraction; B, washed membrane fraction; C, detergent-solubilized fraction (1.2 g of dodecyl β-d-maltoside per g of membrane protein); D, first DEAE-Sepharose FF eluate; E, second DEAE-Sepharose FF eluate.