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 (Mr = 38 500) contains an FAD and Fe2S2 centre and passes electrons to the hydroxylase (Mr = 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 Å with an average first-shell Fe-O/N distance of 2.05 Å. 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 μ-hydroxo or μ-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 3 bonds on the lefthand side signC-H into 3 bonds on the lefthand side signC-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, 3 bonds on the lefthand side signC.. 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 FeIII-O-O unit or an FeV-O unit.
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