Evidence that a type-2 NADH:quinone oxidoreductase mediates electron transfer to particulate methane monooxygenase in methylococcus capsulatus.
NADH readily provides reducing equivalents to membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) in isolated membrane fractions, but detergent solubilization disrupts this electron-transfer process. Addition of exogenous quinones (especially decyl-plastoquinone and duroquinone) restores the NADH-dependent pMMO activity. Results of inhibitor and substrate dependence of this activity indicate the presence of only a type-2 NADH:quinone oxidoreductase (NDH-2). A 100-fold purification of the NDH-2 was achieved using lauryl-maltoside solubilization followed by ion exchange, hydrophobic-interaction, and gel-filtration chromatography. The purified NDH-2 has a subunit molecular weight of 36 kDa and exists as a monomer in solution. UV-visible and fluorescence spectroscopy identified flavin adenine dinucleotide (FAD) as a cofactor present in stoichiometric amounts. NADH served as the source of electrons, whereas NADPH could not. The purified NDH-2 enzyme reduced coenzyme Q(0), duroquinone, and menaquinone at high rates, whereas the decyl analogs of ubiquinone and plastoquinone were reduced at approximately 100-fold lower rates. Rotenone and flavone did not inhibit the NDH-2, whereas amytal caused partial inhibition but only at high concentrations.
Cook
Scott A
SA
Department of Biochemistry, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9142, USA.
Shiemke
Andrew K
AK
D004579:Electron Transport; D007700:Kinetics; D020619:Methylococcus capsulatus / Q000201:enzymology* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D011808:Quinone Reductases / Q000302:isolation & purification / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D013054:Spectrophotometry, Atomic; D013379:Substrate Specificity
D011809:Quinones; D009243:NAD; D010105:Oxygenases; C021555:methane monooxygenase; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases