ELECTRON-PARAMAGNETIC RESONANCE STUDIES OF THE MECHANISM OF SUBSTRATE OXIDATION BY METHANE MONOOXYGENASE

Soluble methane monooxygenase from Methylococcus capsulatus (Bath) and Methylosinus trichosporium (OB3b) comprises three proteins that are necessary for the formation of methanol from methane, NADH2 and O2. The reductase (M(r) = 38 500) contains an FAD and Fe2S2 centre and passes electrons to the hydroxylase (M(r) = 250 000) which contains a bridged diiron centre. The third protein (protein B) contains no metal ions or cofactors and plays a regulatory role. Various spectroscopic techniques have been used to show that there is a very close similarity between the iron centres in the hydroxylases in which the Fe-Fe distance is 3.41 angstrom with an average first-shell Fe-O/N distance of 2.05 angstrom. The latter values varied according to the oxidation state of the binuclear centre. The data suggest that there is no short Fe-O bond but that a mu-hydroxo or mu-alkoxy bridge may be present. The major role of this enzyme is to convert methane into methanol, but it is quite catholic in its choice of substrates and will convert C-H into C-OH in many systems. We have used a range of spin traps coupled with EPR spectroscopy to show that the first stage of these conversions is to form the carbon-centred radicals, C.. Direct evidence for .CH3, .CH2OH and a range of other radical intermediates have thereby been obtained. There is no evidence for the formation of .OH or related radicals, which rules out one of the possible reaction mechanisms. A detailed scheme for reactions in which C-H is replaced by C-OH is proposed. There is a second type of reaction, typified by the formation of pyridine N-oxide from pyridine, which does not appear to proceed by a free-radical mechanism, since no spin-trapped species were detected. We suggest that these processes involve direct oxygen atom transfer, either from an Fe(III)-O-O unit or an Fe(V)-O unit.