Methane monooxygenase: purification and properties of flavoprotein component.
An anaerobic procedure was developed for the purification of the flavin:NADH oxidoreductase (flavoprotein) component of methane monooxygenase to homogeneity. The molecular weight of the flavoprotein determined by gel filtration was about 40,000, and by sedimentation equilibrium analysis, about 38,000. The purified flavoprotein is a monomeric protein with a sedimentation constant (S20,W) value of about 2.1 S. The absorption spectrum of the flavoprotein has a peak at 460 nm and shoulder at 395 nm. The fluorescent excitation and emission spectra of the fluorescent component of flavoprotein had peaks at 450, 370, and 530 nm, respectively. A FAD was identified as a prosthetic group of flavoprotein by thin-layer chromatography. The flavoprotein contained about 1 mol of FAD and 2 mol each of iron and acid-labile sulfide per mole of protein. The flavoprotein was directly reduced by NADH under anaerobic conditions. The formation of neutral flavin semiquinone was detected during anaerobic titration of flavoprotein by NADH and also as a free radical signal at a g value of 2.004 by EPR spectroscopy. The iron sulfur cluster has g values of 2.04, 1.96, and 1.87, yielding a g average of 1.96, characteristic of a Fe2S2 center. Antibody prepared against the flavoprotein reacted with flavoprotein and inhibited methane monooxygenase activity.
D002850:Chromatography, Gel; D004578:Electron Spin Resonance Spectroscopy; D038181:FMN Reductase; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007159:Immunologic Tests; D007501:Iron / Q000032:analysis; D008772:Methylococcaceae / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013440:Sulfides / Q000032:analysis; D014461:Ultracentrifugation
D013440:Sulfides; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D007501:Iron; D010105:Oxygenases; C021555:methane monooxygenase; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases