FURTHER EVIDENCE FOR MULTIPLE PATHWAYS IN SOLUBLE METHANE-MONOOXYGENASE-CATALYZED OXIDATIONS FROM THE MEASUREMENT OF DEUTERIUM KINETIC ISOTOPE EFFECTS

The data from the deuterium isotope experiments in this study show tha t the primary kinetic isotope effect for methane oxidation catalysed b y soluble methane monooxygenase from Methylococcus capsulatus (Bath) i s very small, <2. In contrast, the primary kinetic isotope effect for -CH3 group oxidation in toluene is large, >7. A mechanistic pathway in which a substrate radical is formed from hydrogen atom abstraction by a ferryl species is believed to operate for CH4, the toluene -CH3 gro up and similar alkanes. Direct oxygen atom addition, rather than H ato m abstract- tion, is indicated for aromatic ring oxidations in benzene and toluene and for styrene oxide formation from styrene. Thus, more than one mechanistic pathway appears to operate in soluble methane-mon ooxygenase-catalysed reactions and, in some cases, the pathway chosen may be dictated by the substrate. In the soluble methane-monooxygenase -catalysed oxidation of toluene the rates of: (a) substrate dissociati on from the enzyme-substrate complex, (b) product formation and (c) pr oduct release (benzyl alcohol and p-cresol) from the enzyme-product co mplex are comparable in magnitude. Therefore all three of these steps are partially rate-determining in the soluble methane monooxygenase ca talytic cycle for toluene oxidation.