Substrate radical intermediates in soluble methane monooxygenase.
EPR spin-trapping experiments were carried out using the three-component soluble methane monooxygenase (MMO). Spin-traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), alpha-4-pyridyl-1-oxide N-tert-butylnitrone (POBN), and nitrosobenzene (NOB) were used to investigate the possible formation of substrate radical intermediates during catalysis. In contrast to a previous report, the NADH-coupled oxidations of various substrates did not produce any trapped radical species when DMPO or POBN was present. However, radicals were detected by these traps when only the MMO reductase component and NADH were present. DMPO and POBN were found to be weak inhibitors of the MMO reaction. In contrast, NOB is a strong inhibitor for the MMO-catalyzed nitrobenzene oxidation reaction. When NOB was used as a spin-trap in the complete MMO system with or without substrate, EPR signals from an NOB radical were detected. We propose that a molecule of NOB acts simultaneously as a substrate and a spin-trap for MMO, yielding the long-lived radical and supporting a stepwise mechanism for MMO.
Liu
Aimin
A
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Jin
Yi
Y
Zhang
Jingyan
J
Brazeau
Brian J
BJ
Lipscomb
John D
JD
D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism*; D020602:Methylosinus trichosporium / Q000201:enzymology; D009603:Nitroso Compounds; D010105:Oxygenases / Q000502:physiology*; D012995:Solubility; D019097:Spin Trapping
D005609:Free Radicals; D009603:Nitroso Compounds; D010105:Oxygenases; C021555:methane monooxygenase; C012456:nitrosobenzene