Evidence That a type-2 NADH:quinone Oxidoreductase Mediates Electron Transfer to Particulate Methane Monooxygenase in Methylococcus Capsulatus

Arch Biochem Biophys. 2002 Feb 1;398(1):32-40. doi: 10.1006/abbi.2001.2628.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Electron Transport
  • Kinetics
  • Methylococcus capsulatus / enzymology*
  • Methylococcus capsulatus / metabolism
  • NAD / metabolism
  • Oxygenases / metabolism*
  • Quinone Reductases / isolation & purification
  • Quinone Reductases / metabolism*
  • Quinones / pharmacology
  • Spectrophotometry, Atomic
  • Substrate Specificity

Substances

  • Quinones
  • NAD
  • Oxygenases
  • methane monooxygenase
  • NADH dehydrogenase (quinone)
  • Quinone Reductases